Use of tree sap to preserve sperm cell lines

ABSTRACT

A method of cryogenically preserving sperm comprising (a) combining sperm to be cryogenically preserved and a composition that comprises (1) a cryoprotectant, comprising one or more tree saps; and (2) an extender medium to produce a sperm/medium combination; and (b) subjecting the combination to conditions that result in cryopreservation of sperm, thereby producing a cryopreserved combination that comprises cryopreserved sperm is disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 15/569,164 filed on Oct. 25, 2017, which represents the national stage entry of PCT International Application No. PCT/US2016/029351 filed on Apr. 26, 2016 and claims priority to U.S. Patent Application Ser. No. 62/153,197, filed Apr. 25, 2015, the contents of all of which are incorporated by reference as if set forth in their entirety herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

N/A

BACKGROUND

In general, the present invention involves the use of tree sap to cryogenically preserve avian and mammalian sperm cells, preferably for use in the poultry industry, birds of prey preservation, and preservation of endangered or threatened avian species. The present invention may also be used in the cattle industry, pig industry, equine industry, and in mammalian veterinary medicine.

Avian spermatozoa have a shape that makes the spermatozoa hard to freeze. The spermatozoa are long and thin and are shaped like a whip. This makes the cells very subject to cryogenic injury because they have a large surface area that can be damaged easily upon freezing or processing. Mammalian sperm will also benefit from the present invention because even though these cells are easier to freeze, they are still subject to damage from the cryogenic processes. (Reference; Avian Semen Cryopreservation: What Are the Biological Challenges? J. A. Long, 2006 Biotechnology and Germplasm Laboratory, Animal and Natural Resources Institute, Beltsville Agricultural Research Center, Agricultural Research Service, USDA, Beltsville, Md. 20705, 2006 Poultry Science Association, Inc. Accepted Sep. 10, 2005)

Currently, avian spermatozoa are frozen using several techniques. One technique uses the addition of a cryoprotectant to a fluid media that suspends and supports the cells. The first step in the procedure is to collect the semen and then add a liquid extender. A semen extender is a liquid diluent which is added to semen to preserve its fertilizing ability. The extender allows the semen to be freighted to the female, rather than requiring the male and female to be near to each other. Special freezing extender also allows cryogenic preservation of sperm (“frozen semen”), which may be transported for use, or used on-site at a later date.

This extender/cell mixture is then placed in a refrigerator to chill the mixture down to a desired temperature that allows the cells to line up the lipid components in their outer cell membrane prior to freezing. This is a form of “cold acclimation” and helps to allow the cells to survive the cryogenic process. The method also reduces the temperature gradient drop that the cells have to go through before they reach the freezing point and reduces the cell damage when being frozen.

Once the cells are chilled/acclimated, the cryoprotectant is added to the extender/cell mix, the mixture is packaged quickly and either flash frozen by quick immersion in the liquid nitrogen, pelletized and flash frozen and then packaged into cryo-vials, or suspended above the liquid nitrogen in the vapors to freeze more slowly before it is immersed in the liquid nitrogen. Both fast and slow freezing can be done based on species requirements. Different cryoprotectants that are added to the mix commonly include DMSO (Dimethyl sulfoxide), MA (Methyl-Acetamide), and DMA (Dimethyl Acetamide). These chemicals act as intracellular cryoprotectants while the non-cell wall-permeable chemicals act as extracellular cryoprotectants. These are also known to damage the cell wall during cryopreservation and this impairs fertility.

A better and more effective way of preserving avian and mammalian semen is needed in the art.

SUMMARY OF THE INVENTION

In one embodiment, the present invention is a method of cryogenically preserving sperm comprising (a). combining sperm to be cryogenically preserved and a composition that comprises (1) a cryoprotectant, comprising one or more tree saps; and (2) an extender medium to produce a sperm/medium combination and (b). subjecting the combination to conditions that result in cryopreservation of sperm, thereby producing a cryopreserved combination that comprises cryopreserved sperm. In one version of the invention the sperm is avian sperm. In one version the sperm is derived from the Northern goshawk (Accipiter gentilis).

In another version of the invention the sperm is derived from a mammalian non-human species, preferably selected from the group consisting of cattle, pigs and equines.

In one version of the invention the sap is either maple tree sap or birch tree, preferably both first run saps.

In one version, the present invention is the cryopreserved combination resulting from the method described above.

In another version, the present invention is a method of fertilizing an egg cell comprising the step of introducing the combination described above to an unfertilized egg cell, wherein the egg becomes fertilized.

DESCRIPTION OF THE INVENTION

In general, the present invention is a method and medium useful for the cryogenic preservation of sperm using tree saps. In another embodiment, the present invention is a composition comprising the mixture of the preservation medium and the sperm, using tree saps.

Although the present invention is useful for all animal sperm, the invention is most preferably used with avian sperm because of the special physiologic needs of the avian samples. Preferred avian species include birds of prey (such as Falconiforms and Strigiformes) and commercial species such as turkeys, chickens (Galliformes) and ducks (Anseriformes.) Other preferred avian species include but are not limited to Passeriformes and Psittacifomes.

In another version of the invention, one may wish to preserve the sperm of other mammalian species, including cattle (Family—Bovidae), pigs (Family—Suidae), horses (Family-Equidae) and veterinary medicine applications, including canine (Canidae) and feline (Felidae) species.

In certain embodiments, the method of cryogenically preserving sperm comprises: (a) combining sperm to be cryogenically preserved with a medium comprising (1) a cryoprotectant, such as one or more tree saps or its extracts; and (2) an extender designed to support cell life, wherein the combination produces a sperm/medium combination (cryoprotective medium/sperm combination); and (b) subjecting the combination to conditions that result in cryopreservation of sperm, thereby producing a cryopreserved combination that comprises cryopreserved sperm.

The typical sperm extender typically contains chemicals to both stabilize and protect cell membranes. The Examples below use Beltsville Turkey Extender (BTE) recipe with the exception of removing fructose as one of the ingredients. The fructose was replaced with sucrose and constitutes a separate extender recipe, also a preferred embodiment of the present invention. It was found that goshawk semen did not do well with fructose as its energy source when being cryogenically preserved. This observation is true for other animal cell lines.

The preferred extender recipe for goshawk semen, and other typical avian semen samples, consists of;

Potassium Diphosphate 3H2O 12.7 grams Sodium glutamate 8.675 grams Sucrose to replace Fructose (Anhydrous) 5.000 grams Sodium Acetate 3 H2O 4.255 grams TES 1.95 grams N-tris Hydroxymethyl Methyl-2Amino- ethane Sulfonic Acid Potassium citrate .64 grams Potassium Monophosphate .65 grams Magnesium Chloride .338 grams Purified water 1022 ml is added to the dry ingredients.

This constitutes a full recipe of the preferred extender for goshawk semen for cryopreservation. The sucrose is often left out of this recipe and supplied just through the addition of tree saps that naturally have sucrose in them. Other base recipes may be preferred for other cell lines in other species to meet those species specific requirements. The sugar supplied for the recipe may come from the sap as in the Examples list in the Excel Spreadsheet.

The present invention involves the use of tree sap as a cryoprotectant. Tree sap is a fluid transported in xylem cells (tracheids or vessel elements) or phloem sieve tube elements of a plant. Two kinds of sap are defined as either Xylem sap or Phloem sap. We include both kinds of sap in our definition.

Tree sap is produced at a time of the year when the trees are going through cold stress and freezing in the temperature ranges that are most harmful to the cells that we are trying to freeze. The trees survive temperatures from freezing to minus 60° F. The trees also survive the daily shift in temperatures that the tree can survive both well above freezing to well below it. The tree sap contains properties that allow it to support cell life even when frozen and when going through rigorous freeze thaw cycles and daily temperature extremes. It contains various sugars, antifreeze proteins, carbohydrates, minerals, phenolic compounds, and other compounds that provide cryoprotective properties. Some of these compounds have yet to be described.

The tree species that are most useful in this invention includes the cold-hardy maple tree species, birch tree species, poplar tree species, aspen tree species, and other trees that can be tapped or where chemicals or fluids can be extracted from them. Tree species from the higher latitude deciduous forests are included even if not listed directly herein.

A common factor in these trees is the amount of sugar in the sap. Sugars have cryoprotective properties. Some avian extender recipes often call for 0.5% of either sucrose or fructose. Most tree species meet or exceed this percentage sugar requirement. Maple tree range anywhere from approximately 0.5% to 4.0% sucrose. Another common factor that these trees have is that they have non-sugar cryoprotectant chemicals in their sap. These chemical may provide stronger cryoprotective properties than that the simple sugars that are easily measured.

There are over 128 species of maple trees worldwide. The sugar maple (Acer saccharum) and black maple (Acer nigrum) produce the most sugar in their saps. The red maple (Acer rubrum) and the silver maple (Acer saccharinum) produce less sugar but are in the latitudes where they will likely contain similar cryoprotective properties in their sap. These later two species are one preferred version of the present invention due to the lower sugar content and potentially higher cryoprotective properties in the sap that are not from sugars.

Birch tree (Family-Betulaceae, Genus-Betula), poplar tree (Family Salicaceae, Genus-Populus), and aspen tree (Family-Salicaceae, Genus-Populus) species come from higher latitudes in the United States and Canada and have lower sugar content in their sap than the maple (Acer species) tree species do. The non-sugar cryoprotective chemicals in their sap will likely be higher as these species survive in a more extreme environment with temperature ranges fluxuating widely below freezing, and the trees have a lower content of the sugars that are known to be cryoprotective in their sap.

Twenty three species of trees that can be tapped in the United States and are useful in the present invention include but are not limited to Sugar Maple (Acer saccharum), Black Maple (Acer nigrum), Red Maple (Acer rubrum), Silver Maple (Acer saccharinum), Norway Maple (Acer platanoides), Boxelder (Acer negundo), Bigleaf Maple (Acer macrophyllum), Canyon Maple or Big Tooth Maple (Acer grandidentatum), Rocky Mountain Maple (Acer glabrum), Gorosoe (Acer mono), Butternut or White Walnut (Juglans cinerea), Black Walnut (Juglans nigra), Heartnut (Juglans ailantifolia), English walnut (Juglans regia), Paper Birch (Betula papyrifera), Yellow Birch (Betula alleghaniensis), Black Birch (Betula lenta), River Birch (Betula nigra), Gray Birch (Betula populifolia), European White Birch (Betula pendula), Sycamore (Platanus occidentalis), Acer ginnala, and Ironwood or hophornbeam (Ostrya virginiana).

Preferably, one would begin with extender recipes designed for the preservation or storage of animal sperm. Typically, the initial amount of sap added to the modified extender recipe will make the initial solution physiologically close to the osmolality of the raw semen and still provide for cryo-protection of the cells. The initial osmolality range needed is determined by the measurement of the osmolality of the raw semen.

Goshawk semen has an osmolality of about 341 mili-osmoles. Later additions of extender/sap combinations increase the osmolality of the mixture to dehydrate the cells immediately prior to freezing. Dehydrating the cells just prior to freezing them, increases survival.

An ideal osmolality level is determined by the end results of the survival of the cells in question and the ability of the stored sample to create fertile female gametocytes. It is known that different species of birds have sperm cells that tolerate different osmolality extremes. Some avian spermatozoa survive very high osmolality and others do not. [See Species Variation in Osmotic, Cryoprotectant, and Cooling Rate Tolerance in Poultry, Eagle, and Peregrine Falcon Spermatozoa; Juan M. Blanco, George Gee, David E. Wildt, and Ann M. Donoghue; Biology of Reproduction Oct. 1, 2000 vol. 63 no. 4 1164-1171] The use of the sap allows both the removal of other toxic cryoprotectants from the mix and/or reduces the amount of toxic cryoprotectants used. Yet, one may still wish to add additional cryoprotectants to the mix.

A typical sperm/sap-extender combination of the present invention is as follows: The final volume of the sperm/sap-extender combination should be no more than 1:3 dilutions making the semen a quarter of the final volume. Semen dilutions higher than this can impair fertility because of simple dilution.

In a preferred version of the invention, sap comprises at least 30% of the final sperm/extender combination. In another version of the invention, sap comprises 10%-80% of the final sperm/extender combination, preferably about 50%.

Over dilution reduces sperm fertility of the sample. The amount of sap needed to provide cryogenic protection to the mixture varies considerably because of tree species variation in cryoprotectant types and properties.

I have been modifying the extender recipe enough to allow sap to be blended into the mix so that this mix then supports the cells when they are frozen in liquid nitrogen (LN₂) with or without the use of an additional cryoprotectant. A typical example of extender includes the dry ingredients of the Beltsville Turkey Extender without the fructose (BTE minus fructose) as a base recipe to work with.

The sap from the Maple tree and the Birch tree were then added to the BTE minus fructose and used at different ratios, to preserve the sperm cells. A recipe that preserved the cells in LN₂ well included 1 part raw semen, 1 part BTE minus fructose with 0.5% sucrose added back in, 2 parts BTE minus fructose with sap added, to supply its liquid volume. The sperm and the BTE minus fructose with 0.5% sucrose added back in; were mixed in a 0.5 ml Eppendorf vial in the fridge. A matching volume of BTE minus fructose with sap as its liquid diluent was also placed in the fridge but in a separate tube. Both tubes were allowed to equilibrate to an equal temperature for 10 minutes before they were then mixed, packaged, and then flash frozen. The work was done in the fridge at 42° F. so there were no temperature fluxuations to stress the semen. This form of cold “acclimation” allowed the lipid component of the cell wall to line up prior to freezing to help prevent damage to the cell structure.

Therefore, a preferred version of the present invention comprises a composition, wherein 1 part of the volume is raw semen; 1 part of the volume is extender, such as BTE no fructose plus 0.5% sucrose added back in; and 2 parts of the total volume was BTE no fructose with sap of either the maple or birch tree. A minimum of 50% sap by volume should preferably be used in this mix. Samples with 50% sap by volume had far better survival on thaw than samples with less than this percentage.

The packaging consisted of the semen being placed in a 75 ul Mylar coated capillary tube with one end being caulked. Its opposite end was left open. This capillary tube was then placed inside a standard plastic poultry straw and the end opposite of the cotton was crimped shut. The poultry straw was then placed inside a plastic soda straw that had holes cut in the side of it. These holes allowed the LN₂ to enter and surround the poultry straw quickly as it was immersed. The holes in the soda straw also allowed the package to drain and breathe as it was thawed so that it did not explode.

Cryopreservation can be carried out at any time after production of the medium/sperm combination as long as the storage does not significantly adversely affect the viability of the sperm. For example, cryopreservation can often be carried out as long as 180 minutes after the sperm/medium combination is produced with no loss of fertility. Samples should be chilling to extend the shelf life before freezing. A typical temperature for storing avian semen at is 5° C. Chilling the semen helps to line up the lipid component in the cell wall prior to freezing. This increases cell survival.

Preservation is typically carried out at a temperature minus −198° C. In specific embodiments, cryopreservation is carried out at a temperature between from about minus −80° C. to about minus −198° C. In one preferred embodiment, the cryo-protection takes place in a liquid nitrogen bath/canister and the vials are stored at a −198° C. Long term storage can be achieved by placing the storage vials or straws in a liquid nitrogen canister. One would then wish to use the preserved sperm to fertilize a female gametocyte, female germ cell or ovum.

Before the sperm is used for artificial insemination or incubated with a female gamete, the sperm is typically thawed and may also be washed. Sperm samples are often thawed in cold water or warm water baths with the temperature requirements being determined both by the species cell requirements or the cryoprotectant type used in the mix. Avian spermatozoa are typically thawed in ice water baths or cool water baths, and bovine spermatozoa are typically thawed in warm water baths that are body temperature. Insemination is performed immediately after thaw. Sperm are sometimes concentrated into pellets with the contents of different straws being combined, centrifuged down, to form a pellet of semen.

In all embodiments described herein, the resulting cryopreserved sperm can be stored indefinitely.

The fertilization capacity or ability of sperm can be assessed using methods known to those of skill in the art, such as in vitro methods, including assessing the ability to fertilize the oocytes/female gamete with which they are combined/incubated (their ability to form-cell embryos, for example) and/or in vivo methods, including assessing the production of offspring by females into whom the fertilized oocytes/female gamete are implanted (mammals). Fertilization capacity or ability can be assessed using available methods, such as a functional assay, including, but not limited to, a motility assay, a viability assay, a hemizona assay (binding of the sperm to the zona pellucida) or sperm penetration into zona-free mammalian or avian oocytes.

The commercialization of cryogenically freezing avian semen has eluded scientists for decades. The freezing process has not been successful enough. Current papers cite approximately 35 to 40% semen motility after thaw. See; Comparative cryopreservation of avian spermatozoa; Benefits of non-permeating osmoprotectants and ATP on turkey and crane sperm cryosurvival. By Juan M. Blanco, Julie Long, George Gee, David E. Wildt, Ann M. Donoghue, Received 24 May 2010 Accepted 10 Dec. 2010. Elsevier B. V.

The present invention, comprising the improvement of using sap as the sole cryoprotectant, often showed greater than 50% survival based on Live/Dead stains done after the thaw of samples. Some examples showed up to 73% survival on thaw with no additional cryoprotectant being used. Once this successful idea is combined with the other currently successful ideas of science, the survival of the semen will likely be high enough to make avian semen cryopreservation a commercially viable venture.

Other animal species will likely benefit from this invention as well. There are numerous articles on scientists trying to freeze the semen of other animal species with limited success. The use of tree sap harvested at winter's first thaw, and used in cryopreservation of cell lines is an exciting and now documented success. The success of this process must also be evaluated based on the improved fertility and hatchability of eggs produced from females inseminated with frozen semen.

EXAMPLES

In general, the present invention involves the use of tree sap to cryogenically preserve avian sperm lines, preferably for use in the poultry industry, birds of prey preservation, preservation of endangered or threatened avian species, and other avian species. It will also be useful in pigs (Family-Suidae), cattle (Family-Bovidae), horses (Family-Equidae), dogs (Family-Canidae), and cats (Family-Felidae).

Table 1 contains the results of many experimental trials. In general, I obtained maple tree sap from the native trees in southeastern Wisconsin. The osmolality of Maple tree #3 is 100 mili-osmoles.

The raw dry ingredients for the preferred medium were as follows: Beltsville Turkey Extender recipe, minus the fructose; had maple tree or birch tree sap added for a final volume of 100 ml. (I added 90 ml of sap to make the final volume of the dry and wet ingredients total 100 ml.)

I began with a set of dry ingredients that was for 1/10^(th) of the standard recipe listed above. I added 90 ml of maple tree sap to one jar and 90 ml of birch tree sap to another jar to make a final volume of 100 ml in each jar. No other cryoprotectant was added into the mix. The fructose had been removed so the energy source for the semen came from the sucrose that was already in the sap. The cells that I am trying to preserve do not appear to metabolize fructose well and need the sucrose in the recipe to survive the freezing.

The sap was used full strength in the stock jars, but it was used in different ratios when it was added to raw semen. Sometimes a 1:1:2 dilution was used (1 part semen: 1 part BTE no fructose, plus 1/% sucrose: 2 parts BTE plus Sap); sometimes a 1:1:1 dilution was used. Sometimes a 1:1:1 dilution was used where the final mix was 33% Semen and 66% sap with sap being added into the both the base mixture and the final mixture before freezing. In all cases no other cryoprotectant was added to the sample and only the sap was used to preserve the cells in the liquid nitrogen. The cells survived in large percentages even when no additional (penetrating or non-penetrating) cryoprotectant was added to the mix.

Experiments were also done using the sap from Alaskan birch tree. Again, the raw dry ingredients for the Beltsville turkey extender, minus the fructose ( 1/10^(th) volume of the standard recipe) had birch tree sap added for a final volume of 100 ml. [The recipe for a standard liter volume of BTE is listed above.] No other cryoprotectant was added into the mix.

The maple tree sap had been stored in 100 ml plastic bottles, with about 16 bottles per cardboard box, with the top left open. The top of the bottle had the minerals and other chemicals forced out of it leaving the ice crystals at the top. The center of the bottle had not frozen, and it remained in an almost glass like state without freezing completely after 24 hours. This type of freezing is critical to success when doing cryogenic freezing. This prevents ice crystal formation that damages the cells. [Reference; Investigation of Chemical and Physical Properties of Southwestern Wisconsin Maple Syrup; By Hiroyuki Takano, A Thesis Submitted in Partial Fulfillment of the Requirements for the Master of Science Degree with a major in Food and Nutritional Sciences. Martin G. Ondrus, Thesis Adviser; the Graduate School University of Wisconsin-Stout, December 2005]

The sap of the birch tree was obtained from Alaska through a syrup company called Alaska Wild Harvest LLC, dba Kahiltna Birchworks, PO Box 2267, Palmer, Ak. 99645. I obtained both the first run and second run saps for experimentation. This sap contains three times less sugar on average, than Maple tree sap. This tree comes from higher latitudes that are subject to more severe temperatures and temperature swings than the forest in Wisconsin are. The birch tree sap froze very slowly in the chest freezer and in a similar manner to that of the maple tree sap listed in the previous paragraph.

The first semen sample that I froze was from a male Northern goshawk (Accipiter gentilis) using an extender recipe that was modified to include maple tree sap. This recipe consisted of all of the dry ingredients of the Beltsville Turkey Extender in the usual percentages, without the fructose. 90 ml of maple tree sap was added for a final volume of 100 ml. (This is 1/10^(th) of a standard recipe for BTE) One hundred percent of the liquid added into the recipe was maple tree sap. The sucrose content of the maple tree sap is reported in the literature to be between 2%-2.6%. The exact sucrose level of this sap was not measured but estimated to be about 2% because this was a first run sap. The needed minimum sugar level for the Beltsville Turkey Extender is 0.5%. So this recipe ended up having more sugar in it (than the commercial extender) because the sap had 4-5 times the needed sugar level, naturally in its sap.

This first recipe contained 4-5 times the needed sugar, making it hyperosmolar so that the sperm would gradually lose motility at room temperature. However, a frozen semen sample (Sample #69, Table 1) from the Northern goshawk (Accipiter gentilis) was immediately flash frozen upon mixing with the extender-sap combination 1:2 (1 part semen and 2 parts Extender/Sap combination) and 58% of the cells survived the freezing and thawing process based on a live/dead stain (Eosin/Nigrosin) and visual observations. This sample was thawed in a cool water bath at approximately 55° F. after being in the liquid nitrogen can for over a day. These cells then went on to lose motility at nearly at exactly the same rate as a sample that had been mixed and held at room temperature due to the chemical makeup of the sample. However, the cells survived the freezing process essentially unchanged. The motility and linear movement of the cells was left nearly intact, being unaltered by the freezing process. This was my first documented success and it exceeded my expectations. Cell survival post freezing showed great success.

There was no other cryoprotectant put into the sample. This recipe is clearly hyperosmolar (and detrimental to the cells) because it contained at least 2% sucrose and the sperm only needed 0.5% sucrose.

I found 58% survival upon thaw based on a live/dead Eosin/Nigrosin stain on this first sample. A hundred cells were counted using a standard lab cell counter and this simple percentage established. This exceeded literature references of 25% with standard cryoprotectants such as DMA and MA. A survival rate of above 25%, preferable above 40% or 50%, indicates a successful experiment.

A second sample of 22 ul semen (sample #81, Table 1) was then frozen. 22 ul Beltsville Turkey Extender, no fructose, plus 0.5% sucrose was added to the semen in a 0.5 ml Eppendorf tube and placed in the fridge. 44 ul of BTE with maple tree sap; was placed in its separate tube, also in the fridge at 42° F. The two liquids were combined after acclimating in the fridge for 10 minutes. The total volume was 88 ul. The sample was packaged in 2-75 ul Mylar coated capillary tubes, placed in poultry straws, this was then placed in a ventilated soda straws, and flash frozen. Both straws were thaw in a 55° F. water bath. Two straws were produced from one semen sample. Sample 1 had 25-30% live forwardly motile sperm with normal speed of travel and a live/dead stain of 50 live/50 dead. The second straw had 55% forwardly motile with normal motility and a live/dead stain of 57 live/43 dead. The semen survival increased when the percentage of the Maple sap was lowered to 50%.

Additional samples of semen from this male goshawk were frozen. The results are listed in Table 1, a table disclosing semen samples where either maple sap or birch sap were used exclusively for cryopreservation. The semen of the Northern goshawk (Accipiter gentilis) was used in all experiments.

I list samples in Table 1 that are successful and those that are not in the column marked “Is this sample workable?” Samples were listed as Yes, No, and Maybe. There is a column that lists the success or lack of success; so it is easy to review the table quickly by looking down this single column. I had success freezing samples in LN₂ as soon as I started to add the natural Maple tree sap into the formula. Semen cells survived cryogenic freezing when only tree sap was used as the cryoprotectant, even when there was no other chemical cryoprotectant used. My samples survived the trauma of freezing almost as if they had never been frozen; continuing to swim at a normal speed in a straight direction. The cells eventually lost motility due to problems associated with the solution that they were put in.

It is of particular note that some of the samples survived with even higher survival percentages and motility without the addition of other cryoprotectants, where only the sap was used. Sample 84 survived the best and is nearing the noted highest percentage of survival and motility known to scientists that work in this field of cryopreservation at 73 L/27 D % live/dead stain and a second sample with a 64 L/36 D % live/dead stain. Samples 67, 69, 70, 71, 72, 80, 81, 84, 93, 97, 118, 120, 126, and 128 show very encouraging results with progressive forward motility and the live dead stain percentages listed in the chart above. Other samples also showing this trend are also listed in the table below.

Percentage Survival on Live/Dead Stain Sample numbers in Table 1  0-4% Survival 106, 113, 116, 117  5-9% Survival 82, 100, 101, 105, 109, 110, 115, 116 10-14% Survival 82, 88, 95, 105, 107, 110, 111, 111, 113, 114 15-19% Survival 87, 88, 93, 100, 107, 108, 109, 115 20-24% Survival 82, 91, 100, 102, 103, 106, 108 25-29% Survival 99, 99, 114, 116, 117 30-34% Survival 72, 77, 83, 89, 90, 90, 102, 109, 109, 119, 119 35-39% Survival 44, 77, 77, 85, 91, 92, 99, 111 40-44% Survival 68, 85, 80 45-49% Survival 72, 83, 118 50-54% Survival 81, 97, 103, 105, 107, 110, 112 55-59% Survival 69, 80, 81, 93, 93, 128 60-64% Survival 84, 120 65-69% Survival 72, 76 70-74% Survival 84

Some of the samples showed quiescense features and according to a live/dead stain, survived the freezing but were not motile. Live cells do not take up Live/Dead stain and show up as white on microscope slides, even when no longer motile. These samples are likely not dead and can be“resurrected” and made motile with known techniques. Many of the samples had cells with near normal gross cellular features post freezing and did not appear to be distorted or damaged from the freezing process; on the live/dead stains. These stains have been retained for future reference.

The sap is the key ingredient for cryopreservation because it is non-toxic, has no contagious agents to transmit to the sperm, is plentiful, has key cryo-preservative properties, is in a liquid state naturally, can be collected without bacterial contamination, and it is not viscous (thick) so it does not impair spermatic motility through the fluid medium. It is a natural product that is very unlikely to contain adulterant chemicals.

I envision typical optimizations of the present invention. First, the optimized-liquid base that supports the cell lines needing to be preserved will need to be developed and then modified to allow the addition of the sap to the mix in various percentages, so that the sap does not add chemicals in concentrations that would then kill the cells, but would still allow for cryo-protection. (For example, the osmolality of maple sap that I obtained was 100 mili-osmoles.) This osmolality appeared to be too high when it was added directly to Beltsville Turkey Recipe dry ingredients that did not have the fructose added into the recipe. The cells survived the freezing in great shape, but lost motility possibly due to the hyperosmolality of the approximately 2% sucrose in the maple sap.)

Second, the sap ingredient may be optimized just by choosing different species of trees to use. The sugar content in the saps varies with the tree species and so do the other chemicals that act as natural cryoprotectants that are not sugars. Syrup producers use maple trees that produce the most sugar and some syrup producers use birch trees for this process. They know that where maple tree sap is boiled down, between 20-50 units per one unit of syrup is required. When birch tree sap is boiled down, 150 units per 1 unit of syrup is required. Syrup producers do not tend to use maple tree species that produce low sugar content in their sap. Yet, these trees also survive the rigorous temperatures and temperature extremes and must be adapted well to survive without sugar as a main cryoprotectant, implying that other chemicals in the sap that are not sugars, are acting in this manner.

Additionally, one might wish to use a combination of saps. Combinations of the maple and birch sap recipes were used in experimentation listed in Table 1. High success rates were achieved with this combination.

Additionally, sap taken at different times during the tapping process may yield some beneficial results. Later run saps are lower in the sugars seen in the earlier run, while the trees are still going through and surviving extreme low temperature stresses. The osmolality of the saps taken at different times may be of benefit.

Additionally, extracts of the saps may yield benefits through the discovery of newly discovered antifreeze proteins or compounds that would be of use with this process. [Reference; When plant cells can survive ultra-low temperatures; Pawl M. Pukacki, Physiology of Abiotic Stress Laboratory, Institute of Dendrology, Polish Academy of Sciences, Kornik, Poland].

By “sap,” we mean to include any concentration or dilution of tree sap. For example, the cryoprotectants in the sap can be concentrated. One preferable way of concentrating the sap is via reverse osmosis. This is a mechanism whereby the water is removed from the sap and the chemicals are concentrated on one side of a semipermeable membrane without having to apply heat to the sap that would likely destroy the chemicals that are cryoprotective.

Suitable Extender Recipes for 2015 and Semen Survival Study.

Number 1 recipe; Beltsville Turkey Extender, No fructose plus 90 ml Maple Tree sap, from first run tapping, QS to 100 ml. Dated on bottle Mar. 11, 2015, mixed on Mar. 27, 2015, Initial pH listed as 7.5 and then after being mixed the pH was 6.73 with my meter.

Maple tree sap is 2-2.6% sucrose which is 4-5 times too high and hyperosmolar. BTE normally has 0.5% Fructose in it. However goshawk eggs do not do well with Fructose and must have Sucrose to survive.

Number 2 recipe; Beltsville Turkey Extender, No fructose plus 90 ml Birch tree sap from Alaska, first run sap, QS to 100 ml. dated on bottle Mar. 11, 2015 and mixed on Apr. 11, 2015. Initial pH listed as 7.5 and then after mixing read on my meter as 7.62.

Number 3 recipe; Beltsville Turkey Extender, No fructose plus 0.5% sucrose, plus 16% Methyl Acetamide by weight. The pH was 7.78. Plus 0.2 mg Inositol (should have been 0.02 mg Inositol). It had a total volume of about 10 ml.

Number 4 recipe; Beltsville Turkey Extender, No fructose, plus ½% sucrose. The final pH was 7.36. The water was boiled and probably has a low oxygen tension.

Purdy formulas were a simple addition of maple tree sap, by volume to BTE. The osmolalities were as listed.

BTE, Control, No sap added, 352 mOsm

BTE, 5% Maple tree sap, 339 mOsm

BTE, 10% Maple tree sap, 326 mOsm

BTE, 20% Maple tree sap, 308 mOsm

The osmolality of the 20% Maple tree sap was too low to support the cells due to cell swelling.

TABLE 1 Date Hen Ratio of Is this Glucose Comments on Num- Date Semen Semen was Sire receiving Semen:Ex- sample Level Semen Survival ber was Used collected. donating. semen. tender. workable? in mg/dl and Storage. 4 Mar. 31, Apr. 6, Squirt Juniper 1 to 2 NO Less than 1% motile on 2014 2011 thaw. Most were dead. 5 Apr. 11, Apr. 9, Squirt None 1 to 3 NO Large sample 40-50 ul. 2013 2011 6 Mar. 25, Apr. 21, Squirt None 1 to 2 NO Some urate 2014 2011 contamination. 0% motility on thaw. 7 X Apr. 23, Squirt None 1 to 2 NO 520 Small amount of urate 2011 mg/dl contamination. 0% motility when thawed. On the blood glucose meter it had a 520 mg/dl reading. Impression; There was decreased speed of motility prefreezing. Was the diluent either too thick? 8 Apr. 11, Apr. 11, Squirt None 1 to 2 NO There was a 2 minutes mix 2013 2011 time and it went straight to the tank and was immersed. It was thawed on Apr. 11, 2013 and there was 0% motility 9 X Apr. 15, Squirt None 1 to 4 NO 435 Rare motility upon thaw. 2011 mg/dl Less than 1%. 10 Apr. 11, Apr. 16, Squirt None 1 to 4 NO Heavy urate contamination 2013 2011 so I diluted with extender so the semen survives. O % motility on thaw. 11 Mar. 31, Apr. 16, Squirt None 1 to 1 NO 0% motility on thaw. 2014 2011 12 Apr. 11, Apr. 24, Squirt 1 to 2 NO 2 cells seen moving. 2013 2011 Almost no survivorship 13 Mar. 31, Apr. 25, Squirt None 1 to 2 NO 0% motility on thaw. 2014 2011 14 Mar. 24, Apr. 26, Squirt Juniper 1 to 2 NO 0% motility on thaw. 2013 2011 15 Mar. 24, Apr. 26, Squirt None 1 to 0 NO BG 5 2% Motile questionable 2013 2011 mg/dl, with thawing. retested as 66 mg/dl 16 Mar. 31, Apr. 27, Squirt None 1 to 2 NO 0% motility on thaw. 2014 2011 17 Mar. 24, Apr. 29, Jasper None 1 to 2 NO Dies quickly when viewed 2014 2011 as a wet prep w/o freezing. 0% motility upon thawing. 18 Mar. 26, Apr. 25, Squirt Juniper Unknown NO 1-5% Motility on thaw. 2014 2011 19 Mar. 25, Apr. 30, Squirt Juniper 1 to 2 NO Tail agglutination a 2014 2011 problem. Survives 3-4 hours at room temperatures. Viewed as a good sized sample wet prep prior to freezing.; Upon thawing saw 5% or less moving and est 1% forward motility. Then put this in Juniper. 20 Mar. 25, May 1, Squirt None 1 to 3 NO Viewed prior to freezing, 2014 2011 tail agglutination is a problem, there is decreased survivorship after the extender is frozen in the fridge and used after thawing. Dilution also not 1 to 2. Viewed as a trace sample wet prep. Exploded on thaw. Capillary tube found and trace saw 0% motility. 21 Apr. 11, May 3, Squirt None 1 to 2 NO Good survivorship in 2013 2011 diluent w/o freezing. Survived 1:30 PM to 6:50 PM, some survival at room temperature. When thawed after freezing there was 0% survival. 22 Apr. 11, Apr. 6, Squirt None 1 to 3 NO Some urate 2013 2013 contamination. 0% motility on thaw. 23 Mar. 30, May 4, Squirt Juniper 1 to 2 YES I put this in Juniper at 1 2014 2011 PM. 25% survivorship with good motility upon thawing. A lot of autoagglutination. Extender frozen prior to use in the freezer. Some survival at room temperature 1 PM to 6:50 PM with little forward motility in wet prep. Impressions: Trout #1 seemed better; Extender best used fresh and not frozen; Cells died faster on this slide and there was more agglutination-Stored extender in 5 cc vials and it was colder at time of use. (Important = Cold shock.) 24 Apr. 2, May 14, Squirt Juniper 2 to 5 MAYBE Heavy urate 2014 2011 contamination. On thaw there was less than 5% motility due to trauma from the explosion of liquid nitrogen. Kevin retrieved this from the can. Used in Juniper the day the tank was filled. 25 Jun. 13, Apr. 10, Squirt None 1 to 4 NO Minor urate contamination, 2013 2013 1% good forward motility with a Ice water thaw. 26 Apr. 11, Apr. 11, Squirt None 1 to 3 NO Minor urate contamination, 2013 2013 0% forward motility on Ice water thaw. 27 Jun. 2, Apr. 11, Squirt None 1 to 8 NO 0% motility on cold water 2013 2013 thaw. No cryoprotectant used. 28 Jun. 3, Apr. 12, Squirt None 1 to 4 NO Lost most of the sample. 2013 2013 Unable to evaluate. No survival seen in ones diluted with water. Cold water thaw. 29 Mar. 31, May 7, Squirt Juniper 1 to 3 NO Less tail agglutination with 2014 2011 this formula. More rapid cell death though after only 20 minutes. Maybe 30% survival, 5% forward motility at 1 hour. None alive on wet prep at 6:50 PM, (collected 1 PM), impression, sperm dies fast in this extender. Less than 1% motile on thaw and put in Juniper Mar. 31, 2014. 30 Apr. 14, Apr. 4, Squirt 1 to 2 NO No motility seen on fresh 2013 2013 wet prep. 7 very mobile cells seen, 1 cell was moving fast and then slowed down and stopped. I used Trout #2 plus ¼ tsp Sorbitol and ⅛ tsp Arabogalactin. 31 Mar. 30, May 30, Squirt Juniper 1 to 4 MAYBE Trace sample after 2014 2013 exploding saw some motility 32 X 2014 May 31, Squirt Juniper 16% NO 8 mm of Semen placed in 2013 room temperature Turkey Extender went to the fridge for 30 minutes acclimation time, Added 3 units DMA for a final volume of 50 mm. Quickly placed over liquid nitrogen in under 60 seconds. 33 X 2014 Jun. 1, Squirt Juniper 44% NO 22 mm of Semen was 2013 placed in Turkey Extender at room temperature and placed in the fridge and acclimated for 30 minutes. 3 units/ul of DMA was added. I was placed over the liquid nitrogen in under 60 seconds. It was hung over the vapors for 10 minutes and then flash frozen. 34 Mar. 29, Jun. 2, Squirt Juniper 48% NO 24 mm of Semen was 2014 2013 placed in Turkey Extender at room temperature to a volume of 47 mm and then acclimated in the fridge for 30 minutes. 3 ul of DMA was added quickly and then the sample was hung over the vapor (in less than 60 seconds) for 10 minutes and then immersed in liquid nitrogen. 35 Apr. 3, May 28, Squirt Juniper 20% NO 8-10 mm of Semen was 2014 2013 collected and Turkey Extender was added that was at fridge temperatures. The final volume with DMA was 50 ul/mm. 35 mm of Extender and 5 ul of DMA was used to make it 10% DMA. “No motility” (probably too cold) seen on the smear on the fresh wet prep. When thawed on ice water 5-10% were seen moving. Only 1% with good not great forward motility. There was progressive loss of motility over minutes. (45 minutes). This is the likely sample that went into Juniper on 4/3. 36 X 2014 May 29, Squirt Juniper 8% 4 mm of Semen was 2013 collected and Turkey Extender that was refrigerator temperature was added to a volume of 47 mm. This was left in the fridge at 40 F. from 7 AM to 6:30 PM, 3 ul of DMA was added quickly and in less than a minute it was hung over the vapors for 10 minutes and then immersed. 50% great motility off trace sample seen before adding the DMA. 37 Mar. 26, Jun. 3, Squirt None 8% 4 mm of Semen was 2014 2013 collected and Turkey Extender was added to a volume of 45 mm. It was acclimated for 30 minutes and then 5 ul (10%) DMA was added. It was Flash Frozen. 38 Mar. 26, Jun. 3, Squirt None 8% NO 2 mm of Semen was 2014 2013 collected and Turkey Extender 23 mm was added and it was acclimated in the fridge for 30 minutes. (10%) DMA was added quickly and it was Flash Frozen in under a minute. 39 Mar. 26, Jun. 4, Squirt None 8% NO 4 mm of Semen was 2014 2013 collected and 45 mm of Turkey Extender was added and it was acclimated in the fridge for 30 minutes. 5 ul of DMA was added (10%) and it was Flash Frozen after mixing in under a minute. 40 Mar. 26, Jun. 5, Squirt None 4% NO 1 mm of Semen was 2014 2013 collected and 8 mm of Turkey Extender was added and it was acclimated in the fridge for 30 minutes. 2 ul of DMA was added and it was flash frozen after mixing in under a minute. 41 2014 2013 4 Squirt Juniper YES 4 TUBES THAT WERE SAMPLES PRESERVED IN 2013 FLOATING THAT WERE PUT IN IN THE COMMERCIAL PLASTIC LIQUID TUBES WITH BEADS TO NITOGEN. CAP THE ENDS, SEMEN WAS IN CAPPILLARY TUBES, WERE FOUND FLOATING IN THE LIQUID NITROGEN ON TOP. BUT LOST THEIR LABELS. SOME OF THESE SAMPLES CONTAINED 25% SURVIVAL OF SEMEN SAMPLES AND WERE ACTIVELY MOVING FORWARD. THESE WERE PUT INTO JUNIPER. ONE OF THE RED AND FOUR OF THE GREEN SAMPLES APPEAR TO BE WHAT I USED FOR AI IN JUNIPER. I DO NOT KNOW WHICH TUBE WAS WHICH BUT I DO KNOW THAT I LOST 3 RED SAMPLES ON THAW DUE TO EXPLODING. THE REMAINING SAMPLES DID NOT LEAK IN THE PRIMARY CONTAINER. MOST OF THE GREEN SAMPLES WERE NOT LOST. THE ENTIRE TANK WAS EMPTIED OF ALL OF ITS SAMPLES IN 2014. STARTING OVER. 16 SAMPLES FOR 2013 PUT IN THE TANK. 42 May 2, Squirt 3 mm No urates. Low cellularity. 2014 Semen to 30 units of Turkey extender, plus 1.5 units DMA 43 May 1, Squirt 6 mm Some urate 2014 Semen, contamination. Low diluted to cellularity. 30 mm volume, added 1.5 units DMA 44 Mar. 1, May 3, Squirt NO No urates. Low cellularity. 2016 2014 Mar. 1, 2016 Thawed in an ice water bath. Low cellularity due to males age. Less than 10% motile. Can not do a Live/Dead stain as cellularity is too low. 45 Mar. 29, May 4, Squirt 2 mm of NO No urates. Low cellularity. 2015 2014 Semen Goshawk semen requires plus 18 mm sucrose and not fructose of Turkey to survive freezing!!! This extender to is why these cells survived a total in the Trout Extender #2 volume of and not the Turkey 20 mm. Extender that has the Fructose! You must use Beltsville Turkey Extender minus the Fructose, with sucrose added back in to .5% (½%) 46 May 4, Squirt 6 mm of Low number of urates. 2014 Semen, Low cellularity. plus 19 mm of Turkey Extender, plus 2 ul of DMA. 47 May 5, Squirt 1-40 mm 1 side of the split sample 2014 sample had more urates than the contaminated other, semen in only one with spot on the tube, so urates split separated into ½ to put into 2-20 most urates in 1 tube. Low mm cellularity. samples, plus 28 mm Turkey extender, plus 3 ul of DMA (6%). 48 May 6, Squirt 6 mm of No urates. Low cellularity. 2014 Semen, plus 14 mm of Turkey extender to a total volume of 20 mm. Plus 1.5 ul of DMA, (7%) 49 May 6, Squirt 10-16 mm Many urates. Small 2014 of Semen amount of semen in one plus 47 mm spot. Final volume 50 mm. of Turkey Low cellularity. extender, plus 3 ul of DMA. 50 May 7, Squirt 6 mm of No urates. Low cellularity. 2014 semen plus 19 mm of Turkey extender plus 2 ul of DMA. 51 May 7, Squirt 6 mm of No urates. Low cellularity. 2014 Semen Lots of blast cells seen. plus 19 mm Goshawk semen requires of Turkey sucrose and not fructose extender to survive freezing!!! This plus 1.25 ul is why these cells survived of DMA in the Trout Extender #2 (5%). and not the Turkey Extender that has the Fructose! You must use Beltsville Turkey Extender minus the Fructose, with sucrose added back in to .5% (½%) 52 Mar. 29, May 9, Squirt None 5 plus mm NO Watery urates seen in 8 of 2015 2014 of Semen 13 mm total initial semen and about volume so had about 5 8 mm of mm of semen present. Urates, Low cellularity. Goshawk plus 27 mm semen requires sucrose of Turkey and not fructose to extender to survive!! These cells can a total not use fructose and this is volume of why these cells survive in 40 mm plus the Trout #2 extender! 2.7 ul of DMA (6%) 53 May 10, Squirt 16 mm of No urates and cellularity is 2014 Semen low but going up. plus 22 mm of Turkey extender for a final volume of 38 mm, plus 2.23 ul of DMA, (6%). 54 May 22, Squirt 5 mm of No urates and cellularity is 2014 Semen low but going up. plus 15 mm of Turkey extender to a final volume of 2 mm, plus 1 ul of DMA (5%). 55 May 12, Squirt 14 mm of A few urates, but not a lot. 2014 Semen Low cellularity but plus 23 mm increasing. Turkey extender to a total of 47 mm. Plus 2.5 ul of DMA (5%). 56 May 14, May 13, Squirt None 4 mm of No Unknown, No urates. Cellularity is 2014 2014 Semen meter low but climbing. plus 20 mm could not Storage straw did not leak of Turkey read this and the cell survivorship extender number. was feeble. plus 5% Maple Syrup, plus 1 ul DMA (5%). 57 Apr. 4, Mar. 22, Odin None 8 mm of No No semen survived 2015 2015 Semen freezing. The fructose in plus 11 ul the sample is the of Beltsville suspected problem Turkey because it slows the Extender speed of the spermatozoa (unaltered) down by half in fresh Plus 1 ul of samples extended with DMA. Total this TE. The volume cryoprotectant needs to 20 ul. also be looked at. All 2015 samples placed in cold Eppendorf tubes that were already in the fridge. Temperature shock might be present. Might be too cold next to refrigerator coils. Goshawk semen requires sucrose and not fructose to survive freezing!!! This is why these cells survived in the Trout Extender #2 and not the Turkey Extender that has the Fructose! You must use Beltsville Turkey Extender minus the Fructose, with sucrose added back in to .5% (½%) 58 Apr. 18, Mar. 23, Odin None 30 ul of NO All 2015 samples placed in 2015 2015 Semen cold Eppendorf tubes that plus 30 ul were already in the fridge. of Turkey Temperature shock might extender be present. Might be too plus cold next to refrigerator Inositol. coils. Lost sample across Plus 2.6 ul the garage as it exploded. of DMA, 62.6 ul total volume. 59 Apr. 18, Mar. 23, Odin None 30 ul of NO A sample that was still left 2015 2015 Semen in the tube before freezing plus 30 ul had 25% survival based of Turkey on a live/dead stain. All extender 2015 samples placed in plus cold Eppendorf tubes that Inositol. were already in the fridge. Plus 2.6 ul Temperature shock might of DMA. be present. Might be too 62.6 ul total cold next to refrigerator volume. So coils. Less than 1% this is 4% Motile with 55 F. water bath DMA. and hand warming. 60 Apr. 18, Mar. 25, Odin None 12 ul of NO All 2015 samples placed in 2015 2015 Semen cold Eppendorf tubes that plus 24 ul were already in the fridge. of Turkey Temperature shock might Extender be present. Might be too plus cold next to refrigerator Inositol. coils. No survival on Plus 2 ul of thawing. Thawed in a cool DMA. 38 ul water bath at 55 F. and total hand warming. volume. So this is 5% DMA 61 Apr. 18, Mar. 25, Odin None 10 ul of NO All 2015 samples placed in 2015 2015 semen plus cold Eppendorf tubes that 18 ul of were already in the fridge. Turkey Temperature shock might extender be present. Might be too plus cold next to refrigerator Inositol, coils. Thawed in cool plus 2 ul of water bath at 55 F. and DMA To a hand warming. No cell total survival. volume of 30 ul. 6.6% DMA 62 Apr. 15, Mar. 27, Odin None 15 ul of NO All 2015 samples placed in 2015 2015 Semen cold Eppendorf tubes that plus 30 ul were already in the fridge. of Beltsville Temperature shock might Turkey be present. Might be too Extender cold next to refrigerator plus coils. Thawed in cool Inositol, water bath at 55 F. and plus 2 ul of then put on a warming DMA, or plate. Less than 1% 4.25% survival. DMA 63 Apr. 15, Mar. 27, Odin None 20 ul of NO All 2015 samples placed in 2015 2015 Semen cold Eppendorf tubes that plus 37 ul were already in the fridge. of Beltsville Temperature shock might Turkey be present. Might be too Extender cold next to refrigerator plus coils. Thawed in a cool Inositol, water bath at 55 F. No plus 3 ul of cells survived. DMA, 5% DMA 64 Apr. 15, Mar. 28, Odin None 16 ul of NO All 2015 samples placed in 2015 2015 Semen cold Eppendorf tubes that plus 29 ul were already in the fridge. of Beltsville Temperature shock might Turkey be present. Might be too Extender cold next to refrigerator with coils. Thawed in a cool Inositol, water bath at 55 F. No plus 3 ul of cells survived. DMA, 6.25% DMA 65 Apr. 15, Mar. 28, Odin None 14 ul of NO All 2015 samples placed in 2015 2015 Semen in cold Eppendorf tubes that 16 ul of were already in the fridge. Beltsville Temperature shock might Turkey be present. Might be too Extender cold next to refrigerator plus coils. Thawed in a cool Inositol, water bath at 55 F. and plus 2 ul of then put on a warming DMA, plate. No cells survived. 6.25% DMA 66 Apr. 15, Mar. 28, Odin None 13 ul of NO All 2015 samples placed in 2015 2015 Semen in cold Eppendorf tubes that 20 ul of were already in the fridge. Beltsville Temperature shock might Turkey be present. Might be too Extender cold next to refrigerator plus coils. Inositol, plus 2 ul of DMA, 5.7% DMA. 67 Mar. 30, Mar. 29, Odin None 16 ul of MAYBE 5 times This sample was flash 2015 2015 Semen higher frozen with no acclimation. plus 24 ul than BTE When I thawed it, it took of BTE, no normally off living just like the fructose, is so this sample did that was plus Maple is viewed at room tree sap, hyper- temperature. There was no other osmolar. about 11% motility and cryoprotectant less than half of these were moving forward well on thaw. A sample that was left on a slide at room temperature had the mature spermatids stop moving within 5 minutes but the immature spermatids did well and kept on moving. The speed of movement was much better and this is clearly an improvement over the BTE with fructose. I can assume that goshawk semen needs sucrose to survive. The sap is 100 milliosmoles and was added to the dry ingredients of the BTE, no fructose. This added in sucrose at 5 times the percentage needed and made it hyperosmolar. The caused the mature cells to die. The immature spermatids, with immature cell walls, could equalize the osmotic pressure. Maple sap is .5-2.6% sucrose. This is first run sap so it is higher in sucrose than last run. Different maple tree species have different percentages of sucrose. 68 Apr. 15, Mar. 29, Odin None ? NO 6% DMA, No survival 2015 2015 69 Mar. 31, Mar. 29, Odin None 19 ul of MAYBE, Sucrose Most of the sample was 2015 2015 Semen MORE likely 5 lost on thaw. The trace in plus 38 ul SUCCESS times the sample has 66% of BTE plus THAN I what is motility based on visual Maple tree HAD needed, estimates. 25 out of 43 sap, first HOPED so this alive on a swim count with run, tree FOR. sample is the counter. On a live number 3. hyper- dead stain there was Live No other osmolar. 58/42 Dead! cryoprotectant 70 Apr. 15, Apr. 3, Odin None 25 ul of YES, but Sucrose This tube is completely 2015 2015 Semen, can do likely 5 full. 5-10% motility on thaw plus BTE, better. times with normal motility. no fructose, what is Thawed in 55 F. water bath plus 50 ul needed, and then placed on a of Maple so this warm plate. tree sap, sample is No other hyper- cryoprotectant. osmolar. 71 Apr. 15, Apr. 3, Odin None 16 ul of YES Sucrose Thawed in 55 F. water bath 2015 2015 Semen likely 5 and then placed on a plus BTE, times warming plate. 10% no fructose, what is motility at thaw and then plus 28 ul needed, the cells slow their motility of Maple so this to 5% estimated visually tree sap, sample is over 5 minutes. No other hyper- cryoprotectant. osmolar. 72 Apr. 15, Apr. 4, Odin None 13 ul of YES Sucrose Thawed in a 55 F. water 2015 2015 Semen likely 5 bath and then a warming plus BTE, times plate. 20% near normal no fructose, what is motility and motility plus 26 ul needed, estimated visually. of Maple so this Thawed fast! Ventilated Tree sap, sample is soda straw is key. no other hyper- cryoprotectant osmolar. was used. 73 Apr. 15, Apr. 12, Odin None 20 ul of NO Sucrose At room temperature, cells 2015 2015 Semen level slow rapidly on a wet prep, plus 40 ul reads at probably still too of BIRCH 479 mg/dl hyperosmolar. No motility tree sap on a on thaw with 55 F. water Accucheck bath and then a warming glucose plate. meter. 74 Apr. 14, Apr. 12, Odin NONE 42 ul of MAYBE Apr. 14 2015 Sample #1 2015 2015 Semen, Orange straw; Thawed in plus BTE, cold water and then no fructose, placed on a warming plus plate. 5-10% motility of Sucrose, normal looking sperm. The Added in rest are moving a little but 75 ul of not swimming forward. BTE, minus fructose, plus sucrose ½% plus MA (methyacetamide) 75 Apr. 13, Odin 45 ul of 2015 Semen split between 3 straws, BTE no fructose, plus ½% sucrose, plus 84 ul of BTE (same as above) with MA (Methyacetamide) 76 Feb. 28, Apr. 14, Odin None 7 ul of Yes Sample was prepped at 2016 2015 Semen room temperature with with 26 ul all items starting at 70 F. of BTE, no Feb. 28, 2016 Teal Blue fructose, Straw, Not ventilated, plus ½% 80% of cells vibrating, sucrose. 10% moving actively, Plus 2 ul Thawed in a 41 F. water of DMA bath. Live 65/45 Dead cryoprotectant. Stain. Final volume of 35 ul. 77 Feb. 28, Apr. 14, Odin None 55 ul of This is All materials start at room 2016, 2015 Semen one of the temperature (70 F.) and Mar. 1, plus 110 ul first then go to fridge to chill 2016 of BTE, no samples to 41 F. Cold packs were fructose, of NO used to carry to the plus ½% FRUCTOSE garage. sucrose, plus Feb. 28, 2016 Thawed in chilled in DMA to 41 F. water bath to a the fridge test if it is warming plate with Live for 10 the 39/61 Dead. minutes, fructose Mar. 1, 2016 Thawed in a plus 8 ul of or DMA 41 F. water bath to a DMA that is warming plate with 10% causing swimming normally and the 50% vibrating in place. samples to fail. Feb. 28, 2016 Yes, No movement- green tube, Thawed 41 F. water bath. Little cell deformity with a Live 39/61 Dead stain. Sample quiescent. Mar. 1, 2016 Yes, 10% are swimming normally, 50% are vibrating, Thawed in a 41 F. water bath. Live 32/68 Dead stain. 78 Apr. 18, Apr. 15, Odin None 38 ul of Maybe, Acclimated at 41 F. in 2015, 2015 Semen, because fridge with sap separate Apr. 18, plus 75 ul the sap from semen until placed in 2015 of Birch preserves capillary tubes for Sap, BTE, the freezing. Acclimated for 10 no fructose, semen minutes. first run.. when Apr. 18, 2015 Thawed in a 55 frozen. F. water bath. Sample 1 But the showed 5% spermatozoa amount of moving forward normally sap with a live/dead stain of needs to 32/68. be May 18, 2015 Sample 2 had reduced. about 5% moving forward Similar normally with a live/dead response stain of 34/66. to Maple tree sap. The cells survive the freeze but stop moving due to hyperosmolality. 79 Apr. 18, Apr. 17, Odin None 28 ul of Maybe, This was first run Birch 2015, 2015 Semen, because tree sap from Alaska. It Apr. 18, plus 28 ul the sap was acclimated after 2015 BTE, no preserves mixed by only placing it fructose, the between gel packs that plus ½% semen were at 41 F. from the sucrose, when fridge and then it was flash plus 56 ul frozen, frozen. Two straws were Birch Tree but the made from this sample. sap in BTE, amount of One was a yellow, and the no fructose, sap other green, ventilated final needs to soda straws. One tube volume of be had 2-5% forward moving 112 ul with reduced. sperm on visual estimate 1:3 semen Similar with a live/dead stain of to Birch response 21/79. The second tube tree sap to Maple had 0% forwardly moving extender. tree sap. and no live dead stain was The cells done. Thawed in a 55 F. survive cool water bath. the freeze but lose motility due to hyperosmolality. 80 Apr. 18, Apr. 17, Odin None 50 ul of Maybe, Thawed in a cool water 2015, 2015 Semen had Maybe bath of 55 F. Apr. 18, 50 ul of Apr. 18, 2015 First sample 2-3% 2015 BTE, no normal motility, with a fructose, live/dead stain of 57/43.; plus 1/% Apr. 18, 2015The second sucrose sample had 5-10% added moving forward normally together in and a live dead stain of 1 tube. 42/58. Later 100 ul of BTE, no fructose, plus Maple sap was added for a final volume of 200 ul. Making the semen ¼ of the total mix. 81 Apr. 18, Apr. 18, Odin None 22 ul of YES, Thawed in a cool water 2015, 2015 Semen had YES bath of 55 F. Apr. 18, 22 ul of Apr. 18, 2015 First sample 2015 BTE, no had 25-30% normal fructose, motility with a live dead plus ½% stain of 50/50.; sucrose Apr. 18, 2015 The second added to it sample had about 55% placed in 1 normal forward motility tube. Later with a live/dead stain of 44 ul of 57/43. 100 cells were BTE, no counted in each group. fructose, plus Maple tree sap was added for a final volume of 88 ul. 82 May 10, Apr. 20, Odin None 65 ul of Yes, Acclimated at 41 F. in 2015, 2015 Semen, Maybe, fridge with sap separate Feb. 24, plus 65 ul No, No from semen until placed in 2016, BTE minus capillary tubes for Green fructose, freezing. Acclimated for 10 straw- plus ½% minutes. Then flash Holder 5, sucrose. frozen. Ice water thaw, Mar. 2, Later 130 10-15% good motility, Live 2016 ul of BTE Dead Stain 24 live/76 Green minus dead. straw- fructose Feb. 24, 2016 Thawed in a 41 Holder 6, plus Birch F. ice water bath, 2-3% Mar. 2, tree sap moving forward well, pH 2016 first run. 7.0-7.2 with a Live22/78 Green Dead Stain. straw Mar. 2, 2016 Thawed in a Holder 6. 41 F. Ice water bath, Green Straw Holder 6, Rare motile sperm with a Live 10/90 Dead Stain. Cells are very distorted. Mar. 2, 2016 Thawed in an ice water bath at 41 F. No motility and the cells are very deformed. Live 7/93 Dead. 83 May 3, Apr. 20, Odin None 60 ul of No, No Acclimated at 41 F. in 2015, 2015 Semen, Mar. 6, 2016 fridge with sap separate Mar. 6, plus 60 ul Yellow from semen until placed in 2016, of BTE − straw, capillary tubes for Mar. 6, fructose, + water got freezing. Acclimated for 10 2016 ½% into minutes. Then flash sucrose. sample. frozen. Later 60 ul YES May 3, 2016 Thawed in Ice of BTE plus Mar. 6, 2016 water, 2% forward Maple tree Yellow motility, Live dead stain, sap, first straw 39 live/61 dead. run Tree Mar. 6, 2016 Thawed in ice #3. added water at 41 F. 1% Motile for a final and 1% moving in place. volume of Live 33/67 Dead stain. A 180 ul. lot of agglutination. It was exposed to water on thaw because it lost the caulk on the end of the tube. Mar. 6, 2016 Thawed in a ice water bath. 15-20% are motile. Agglutination is present. Live 49/51 Dead. 84 May 3, Apr. 21, Odin None 50 ul of YES, Acclimated at 44 F. in 2015, 2015 Semen had YES, fridge with sap separate Mar. 1, 50 ul of YES from semen until placed in 2016, BTE, no capillary tubes for Mar. 6, fructose, freezing. Acclimated 10 2016 plus 1/% minutes. Then flash sucrose frozen. added May 3, 2015 Thawed in Ice together in water bath, 55%-60% 1 tube. forward motility, Live dead Later 100 stain 39/61. No pH done. ul of BTE, Mar. 1, 2016 Thawed in an no fructose, ice water bath at 41 F., plus Maple Over half are moving sap was forward with fast motility, added for a pH of 7, with a Live 64/36 final Dead stain. volume of Mar. 6, 2016 Thawed in an 200 ul. ice water bath at 41 F. Making the Over 60% are motile with semen ¼ little deformity. They have of the total fast motility with a Live mix. 73/27 Dead stain. 85 Mar. 2, Apr. 21, Odin None 50 ul of Maybe - Acclimated at 44 F. in 2016, 2015 Semen had Quiescent, fridge with sap separate Mar. 2, 50 ul of Maybe from semen until placed in 2016 BTE, no Quiescent capillary tubes for fructose, freezing. Acclimated 10 plus 1/% minutes. Then flash sucrose frozen. added Mar. 2, 2016 Thawed in an together in ice water bath and put on 1 tube. a warming plate. Had 5% Later 50 ul forward motility and a Live of BTE, no 36/64 Dead stain. fructose, Mar. 2, 2016 Yellow straw, plus Maple Thawed in an ice water sap was bath at 41 F. Less than 1% added for a are motile with a Live final 40/60 Dead stain. volume of 150 ul. Making the semen ⅓ of the total mix. 86 May 3, Apr. 21, Odin None 10 ul of No, No acclimation in the 2015, 2015 semen plus Maybe fridge. Flash frozen. May 3, 10 ul of May 3, 2015 First sample 2015 BTE − Fruc, + thawed in ice water, No ½% motility, pH of 7 on pH sucrose, paper, May 3, 2015 Second PLUS sample thawed in ice YOLK, water, 2-5% motility, pH 7 Later 12 ul on paper. Live dead stain of BTE − 48 live/52 dead. Fruc, Plus Maple sap, first run tree 3. 87 Mar. 7, Apr. 22, Odin None 16 ul of Maybe - Acclimated in the fridge in 2016 2015 Semen, Quiescent. separate tubes at 43 F. plus 16 ul The Mixed, packaged, and BTE − Fruc, + cells are then flash frozen. ½% not Mar. 7, 2016 Pink soda straw sucrose, distorted 1% Motile/Cells not PLUS but likely distorted. Live 19/81 Dead YOLK, quiescent stain. Later 16 ul and not BTE − Fruc, motile. plus Maple sap, plus 10% Yolk. Final volume 48 ul. 88 May 3, Apr. 22, Odin None 53 ul of Yes, No Acclimated in the fridge in 2015, 2015 Semen, Thawing separate tubes at 43 F. Mar. 2, plus 53 ul too warm. Mixed, packaged, and 2016 of BTE − No Cold then flash frozen. Pink fructose, + water May 3, 2015 Thawed in Ice straw ½% thaw is no water, 10% moving Holder sucrose, better. forward, pH of 7. Live #6, PLUS dead stain, Live 38/62 Mar. 6, YOLK, Dead. 2016 Later 53 ul Mar. 2, 2016 Thawed in a Pink of BTE − 55 F. water bath, Less than straw fructose, + 1% motile, many cells Holder Maple sap, deformed with a Live #6. plus 10% 16/84 Dead stain. YOLK Mar. 6, 2016 Thawed in an ice water bath, 0% motile, Cells not deformed with a Live 13/87 Dead stain. 89 May 10, Apr. 23, Odin None 45 ul of No, No Acclimated in the fridge in 2015, 2015 Semen with separate tubes at 43 F. Mar. 2, 55 ul of Mixed, packaged, and 2016 BTE − then flash frozen. fructose, + May 10, 2015 A trace sample ½% of this tube survived well sucrose at room temperature. pH and YOLK of 7, Ice water bath thaw, added in at Less than 1% motile, Live 10%, Later dead stain could not be 35 ul of done. Could not read the BTE + Live/Dead stain due to the Birch Tree yolk being present in the Sap so that sample. it becomes Mar. 2, 2016 Orange tube, 26% Birch Thawed in an ice water Tree Sap. bath, Less than 1% motile with a Live 33/67 Dead stain. Many cells are deformed. 90 May 3, Apr. 23, Odin None 45 ul of Maybe, Acclimated in the fridge in 2015, 2015 Semen with Maybe separate tubes at 43 F. Mar. 1, 55 ul of appears Mixed, packaged, and 2016, BTE − Quiescent. then flash frozen. Mar. 2, fructose, + Maybe May 3, 2015 Thawed in a 2016 ½% appears 55 F. water bath. Less than Holder 6 sucrose Quiescent. 1% motile or no motility. Green and YOLK Mar. 11, 2016 Thawed in a ice Straw added in at water bath, No motility but 10%, Later the cells are not deformed 32 ul of with a Live 34/66 Dead BTE + stain = Quiescence. Birch Tree Mar. 2, 2016 Thawed in a ice Sap so that water bath. No motility but it becomes the cells are not deformed 19.7% with a Live 31/69 Dead Birch Tree stain = Quiescence. Sap. 91 Feb. 24, Apr. 24, Odin None 30 ul of Maybe, Mixed at room 2016 2015 Semen, Maybe temperature and then Green with 60 ul appears placed between gel packs soda of BTE − Quiescent. at 43 F. Then flash frozen. straw fructose, + Feb. 24 2016 Green soda from ½% straw from Holder #4 Holder sucrose. Thawed in an ice water #4, Later 30 ul bath at 41 F. Cells not Feb. 24, of BTE − deformed and 2-3% 2016 fructose, motile with a Live 23/77 Deep plus ½% Dead stain. blue sucrose Feb. 24 2016 Deep blue soda plus 18% straw from Holder #4, straw MA (diluted Thawed in an ice water from to a total bath at 41 F., Cells not Holder percentage deformed 80% and 1% #4. of 4.5% motile. But this tube had MA) 10-20% deformed cells overall with a Live 35/65 Dead stain. 92 Feb. 28, Apr. 25, Odin None 15 ul of Maybe Mixed at room 2016 2015 Semen appears temperature and then plus 15 ul Quiescent placed between gel packs of BTE − with 10% at 43 F. The flash frozen. fructose, + movement. Feb. 28, 2016 Thawed in an ½% ice water bath at 41 F., sucrose, ventilated soda straw with plus 15 ul 10% moving forward and a of BTE − Live 35/65 Dead stain. fructose, + Little cell distortion. ½% sucrose plus 18% MA (final of 6% MA). 93 Feb. 24 Apr. 24, Odin None 45 ul of Yes, Yes, Chilled 10 minutes at 2016, 2015 Semen, Yes 45 F. Mixed packaged Green with 45 ul and then flash frozen. straw of BTE − Feb. 24 2016 Green straw Holder fructose, + Holder #4 Thawed in an #4, ½% ice water bath at 41 F., Feb. 24, sucrose, cells not deformed with 2016 Later 45 ul 2-3% motile. Quiescent Green of BTE, − but alive with Live 55/45 straw fructose + Dead stain. Holder ½% Feb. 24 2016 Green soda #4, sucrose straw Holder #4 Thawed in Feb. 24, plus 18% an ice water bath at 41 F., 2016 MA = 6% cells not deformed, 25- Green MA final 30% motile and showed straw concentration slowing motility over 5 Holder minutes with a Live 57/43 #4. Dead stain. Feb. 24 2016 Green soda straw from Holder #4. Thawed in an ice water bath at 41 F. Cells were not deformed and had 5% motile with slowing of motility over 5 minutes. The live/dead stain was hard to read but had Live18/82 Dead. 94 May 10, Apr. 25, Odin None 20 ul of No Chilled for 10 minutes and 2015, 2015 Semen then added DMA. Ice May 10, plus 55 ul water bath thaw. Almost 2015 of BTE − no motility, No live dead fructose, + stain done. Assessment; ½% DMA is not working. sucrose. Later 4.9 ul of DMA added. 95 May 3, Apr. 26, Odin None 22 ul of No, I AM STARTING THE 2015, 2015 Semen Maybe PURDY FORMULAS Mar. 6, plus 66 ul THAT ARE BTE WITH 2016 of BTE + DIFFERENT Orange 10% Maple PERCENTAGES OF straw Sap MAPLE TREE SAP IN PURDY THE DILUTION. THESE FORMULA. HAVE FRUCTOSE AND SUCROSE IN THEM. SEE SHEET ON THESE FORMULAS. May 3, 2016 Ice water bath thaw, 2% moving forward, pH of 7 on paper. Mar. 6, 2016 Thawed in an ice water bath at 41 F. Orange straw with 2% moving forward and 2% moving in place with a Live 14/86 Dead stain. 96 May 10, Apr. 27, Odin None 18 ul of No May 10, 2016 Ice water bath 2015 2015 Semen with thaw, 1-3% of these 54 ul of moving forward. Live dead Purdy 10% stain 31 live/69 dead. Maple tree Comments; Fructose, No sap. 1:3 acclimation, and high pH dilutions. might be a problem. 97 Mar. 6, Apr. 27, Odin None 12 ul of YES! Mar. 6, 2016 No acclimation 2016 2015 Semen and was thawed in an ice Pink soda plus 36 ul water bath. Greater than straw of Purdy 10 50% moving, with 20% Holder #6 % Maple moving normally. There Tree sap. was a Live 52/48 Dead stain. 98 Purdy Purdy Purdy formulas begin with Formulas formulas #95. begin with #95 99 Feb. 24, Apr. 27, Odin None 50 ul of Maybe Processed at room 2016 2015 Semen with sap temperature, placed Holder #5 100 ul of appears between 43 F. gel packs, Pink soda Purdy 10% to and then flash frozen. straw, Maple tree decrease Feb. 24, 2016 Pink straw Feb. 24, sap. movement = Holder #5 Thawed in an 2016 quiescence. ice water bath at 41 F., Holder #4 Maybe = No movement, no pH done, Pink soda Same, Cells very distorted. Had a straw, Maybe = Live27/73 Dead stain. Feb. 24, Same. Feb. 24, 2016 Pink soda straw 2016 Holder #4 Thawed in an Holder #4 ice water bath at 41 F. Pink soda Almost no movement and straw. cells very distorted. Had a Live 29/71 Dead stain. Feb. 24, 2016 Pink soda straw Holder #4 Thawed in ice water bath at 41 F. Less than 1% moving forward and cells were distorted. Live 36/64 Dead. 100 May 16, Apr. 28, Odin None 70 ul of Maybe Processed at room 2015, 2015 Semen Quiescent, temperature, placed Mar. 6, plus 210 ul Maybe between 43 F. gel packs, 2016, of Purdy Quiescent, and then flash frozen. Mar. 7, BTE + 20% No, No May 16, 2015 Thawed in ice 2016 Maple tree water, 1-5% moving well Green sap 308 but motility slows quickly. straw from mOsm. Live dead stain shows 39 Holder #6, live/61 dead, 30 live/70 Mar. 13, dead. Hot plate on 2016 microscope appears to Green speed loss of motility. straw from Mar. 6, 2016 Thawed in an Holder #6. ice water bath at 41 F. 10-15% motility swimming forward. Many of the cells are distorted due to the low osmolality. There is a Live 17/83 Dead stain. Mar. 7, 2016 Green straw. Thawed in an ice water bath. Had 1-2% moving. The cells are very distorted and it had a Live 20/80 Dead stain. Mar. 13, 2016 Thawed in an ice water bath and the cells are very distorted. Less than 1% motility and a Live 9/91 Dead stain. 101 May 3, Apr. 29, Odin None 20 ul of Unknown Mar. 2, 2016 Thawed in an 2015, 2015 Semen as ice water bath at 41 F. Mar. 2, plus 60 ul sample Yellow straw. Cells very 2016 Purdy 10%, from deformed. Live 3/97 Dead Yellow Maple sap holder #5 stain. straw, was lost Holder #6 from straw., Maybe 102 May 10, Apr. 30, Odin None 52 ul of Maybe, Processed at room 2015, 2015 Semen Maybe, temperature, placed Mar. 2, plus 104 ul Maybe- between 43 F. gel packs, 2016 of Purdy Many for 2 minutes and then Orange 5% Maple swollen flash frozen. straw from Sap and May 10, 2015 Thawed in 70 F. Holder #6, distorted water bath, Live dead Mar. 6, cells. stain; 28 live/72 dead, 2016 Mar. 2, 2016 Orange soda Orange straw. Thawed in an ice straw from water bath at 41 F. Had Holder #6. 10% forward motility with many deformed cells. The speed of motility increased with warming. It had a Live 33/67 Dead stain. Mar. 6, 2016 Thawed in an ice water bath at 41 F. It had 10-15% forward motility and many distorted and swollen cells. Live 21/79 Dead. Motility speed increased with warming. 103 May 3, Apr. 30, Odin None 65 ul of NO, No acclimation in the 2015, 2015 Semen, maybe, fridge. Flash frozen. May 10, plus Purdy Yes = Thawed May 3, 2015 in Ice 2016, 10% Maple Quiescent, water, pH 7.5, 0% motility, Mar. 21, Sap, plus Yes = no live dead stain done, 2016 5% (13 ul) Quiescent Sample thawed May 10, 2015 Green of DMA, No Ice water bath, pH 7, 2-3% straw acclimation motility, Live Dead Stain Holder #6, Live 23/Dead 77. Mar. 13, Mar. 2, 2016 Thawed in an 2016 ice water bath at 41 F. Green Saw 2% motile Almost no straw gross cell deformity with a Holder #6 Live 52/48 Dead stain. The longer it warmed on the plate the higher the motility up to 4%. Mar. 13, 2016 Thawed in an ice water bath at 41 F. Less than 1% motility. Little cell distortion and a Live 20/80 Dead stain. 104 May 3, May 1, Odin None 8 ul of No May 3, 2015 Cells do not do 2015 2015 Semen well with this at room plus 8 ul temperature. Thawed in Purdy 10% ice water, less than 2% Maple tree motile, No live/dead stain sap plus done. Arabogalactin, (No MA), Plus Purdy 10% Maple Sap plus Arabogalactin plus 12% MA 105 Apr. 4, Mar. 25, Odin None 55 ul of Maybe; Apr. 4, 2016 Thawed in a ice 2016, 2016 Semen; Mistake water bath. No motility and Apr. 8, plus 55 ul made has a Live 54/46 Dead 2016, of BTE adjusting stain. Apr. 10, with/out pH up in Apr. 8, 2016 Thawed in an 2016 fructose 2nd ice water bath at 41 F. and plus ½% media then palmed to warm. No Sucrose; used - motility with a Live 4/96 plus 55 ul using Dead stain. of BTE w/o Bicarb Apr. 10, 2016 Thawed in an Fruc + 1st changing ice water bath at 41 F. No Run maple the pH motility with a Live 12/88 tree sap, from 6.48 Dead stain. Tree #3. to 7.23. 2015 This appears to impair motility. (Bad), Used 30 drops of Bicarb from a low dose insulin syringe to a 10 cc tube. 106 Apr. 3, Mar. 26, None 50 ul of Maybe; Apr. 3, 2016 Acellular, pH of 2016, 2016 Semen; 50 Mistake 8 on tape, No cells seen Apr. 8, ul of BTE − made on L/D stain. 2016, Fruc + adjusting Apr. 8, 2015 Ice water thaw Apr. 10, ½% Suc; pH up in and then palmed to warm. 2016 Plus 100 ul 2nd pH near 8 on tape. No of BTE − media cells seen, lysed. No L/D Fruc + 1st used - stain. Run Maple using Apr. 10, 2016 Ice water thaw, tree sap. Bicarb pH near 7.5 on tape. No 2015 changing motility seen. Live 21/79 the pH Dead. from 6.48 to 7.23. This appears to impair motility. (Bad), Used 30 drops of Bicarb from a low dose insulin syringe to a 10 cc tube. 107 Apr. 8, Mar. 26, Odin None 47 ul of Maybe; Apr. 4, 2016 Ice water thaw, 2016, 2016 Semen; Mistake pH 7 on tape. No motility. Apr. 4, Plus 23 ul made Live 53/47 Dead stain. 2016, of BTE − adjusting Apr. 8, 2016 Ice water thaw Apr. 8, Fruc + ½% pH up in and then palmed to warm. 2016 Suc; 2nd pH on tape was 7.2. No Plus 65 ul media motility. Live 15/85 Dead of BTE − used - stain. Fruc + 1st using Apr. 8, 2016 Ice water thaw run Maple Bicarb and then palmed to warm. Tree sap. changing pH 7.5 on tape. Live 13/87 2015 the pH Dead. from 6.48 to 7.23. This appears to impair motility. (Bad). Used 30 drops of Bicarb from a low dose insulin syringe to a 10 cc tube. 108 Apr. 4, Mar. 27, Odin None 40 ul Maybe; Apr. 4, 2016 Capillary tube 2016, 2016 Semen; Mistake lost from straw in the tank. Apr. 10, Plus 20 ul made Apr. 10, 2016 Ice water thaw. 2016, of BTE − adjusting No motility. pH of 7.2. No Apr. 10, Fruc + pH up in L/D stain. 2016 ½% Suc; 2nd Apr. 10, 2016 Ice water thaw. Plus 40 ul media pH 7.2 on tape. Less than of BTE − used - 1% motile. Live 24/76 Fruc + 1st using Dead. Run Maple Bicarb Tree #3. changing 2015 the pH from 6.48 to 7.23. This appears to impair motility. (Bad). Used 30 drops of Bicarb from a low dose insulin syringe to a 10 cc tube. 109 Apr. 4, Mar. 27, Odin None 40 ul of Maybe; Apr. 4, 2016 Ice water thaw, 2016, 2016 Semen; Mistake pH 8 on tape. No motility Apr. 4, Plus 40 ul made and a Live 30/70 Dead 2016, of BTE − adjusting stain. Apr. 10, Fruc + pH up in Apr. 4, 2016 Ice water thaw. 2016, ½% Suc; 2nd pH on tape of 7.2. No Apr. 10, Plus 80 ul media motility. Live 30/70 Dead 2016 of BTE − used - stain. Fruc + 1st using Apr. 10, 2016 Ice water thaw Run Maple Bicarb with pH of 7.5 on tape. No Tree #3. changing motility and a Live 19/81 2015 the pH Dead stain. from 6.48 Apr. 10, 2016 Ice water thaw to 7.23. pH of 7.5. This appears to impair motility, (Bad). Used 30 drops of Bicarb from a low dose insulin syringe to a 10 cc tube. 110 Apr. 3, Mar. 28, Odin None 30 ul of Maybe; Apr. 3, 2016 Ice water thaw, 2016, 2016 Semen; Mistake pH on tape 7.2. No Apr. 5, Plus 30 ul made motility. Live 51/49 Dead 2016, of BTE − adjusting stain. Apr. 10, Fruc + ½ pH up in Apr. 5, 2016 Ice water thaw, 2010 % Suc; 2nd pH of 7 on tape. No Plus BTE − media motility. Live 5/95 dead Fruc + 1st used - stain. Run Maple using Apr. 10, 2016 Ice water thaw. Tree sap. Bicarb pH of 7.2 on tape. No 2015 changing motility. Live 16/84 Dead the pH stain. from 6.48 to 7.23. This appears to impair motility. (Bad). Used 30 drops of Bicarb from a low dose insulin syringe to a 10 cc tube. 111 Apr. 4, Mar. 28, Odin None 40 ul of Maybe; Apr. 4, 2016 Ice water thaw, 2016, 2016 Semen; Mistake pH of 7.2 on tape. No Apr. 8, Plus 40 ul made motility and a Live 35/65 2016, of BTE − adjusting Dead stain. Apr. 8, Fruc + pH up in Apr. 8, 2016 Ice water thaw 2016 ½% Suc; 2nd and then palmed to warm. Plus 60 ul media pH of 7.2 on tape. Live of BTE − used - 5/95 Dead. Fruc + 1st using Apr. 8, 2016 Less than 1% Run Maple Bicarb motility on ice water thaw. Tree #3. changing pH 7.2 on tape. Live 7/93 2015 the pH Dead. from 6.48 to 7.23. This appears to impair motility. (Bad). Used 30 drops of Bicarb from a low dose insulin syringe to a 10 cc tube. 112 Apr. 4, Mar. 30, Odin None 42 ul of Yes, one Apr. 4, 2016 Ice water thaw. 2016, 2016 Semen; sample pH of 7.5 on tape. Live Apr. 5, Plus 42 ul did very 25/75 Dead stain. 2016, of BTE, no well and I Apr. 5, 2015 Ice water thaw Apr. 5, fructose + wonder if and then palmed, 50% 2016 ½% Suc it froze motile, pH 7.5 on pH tape, (pH 7.4); more Live 53/47 Dead stain. Plus 65 ul slowly. Apr. 5, 2016 Ice water thaw of BTE − and then palmed to warm. Fruc + plus No motility. Live 8/92 1st Run Dead. Need slow freeze Maple Tree and at least 30% sap for Sap from the cells to survive. pH Tree #3 adjustments bad on (2015), (pH these samples. 7.23) 113 Apr. 4, Mar. 30, Odin None 35 ul of Maybe; Apr. 4, 2016 Ice water thaw, 2016, 2016 Semen; pH no cells seen. No pH Apr. 5, Plus 35 ul adjustment done. No L/D stain. 2016, of BTE − in both Apr. 5, 2016 Ice water thaw. Apr. 8, Fruc + ½% diluents is No motility. No pH done. 2016 Suc (pH stressing Live 19/81 Dead stain. 7.4); Plus the cells Apr. 8, 2016 Ice water thaw. 35 ul of too much pH 7.3. No motility. Live BTE − Fruc + and stops 12/88 Dead stain. 1st run motility. Maple Tree sap tree #3. 2015. (pH 7.23). 114 Apr. 4, Apr. 2, Odin None 25 ul of Maybe; Apr. 4, 2016 Ice water thaw 2016, 2016 Semen; the pH with no motility. pH 8 on Apr. 5, Plus 25 ul and tape. Live 26/73 Dead 2016, BTE − Fruc + speed of stain. ½% Suc freezing Apr. 5, 2016 Ice water thaw (pH 7.4); need to and no motility. pH of 7.4 Plus 35 ul be on tape. Live 10/90 Dead of BTE − changed. stain. Too little sap was Fruc + 1st Slow the used and the freezing Run Maple freeze needs to be slow. pH tree sap down and needs to be lower in (pH 7.23). lower the starting extenders. 2015. No pH to other decrease cryoprotectant. cell metabolism. 115 Apr. 3, Apr. 3, Odin None 65 ul Maybe; Apr. 3, 2016 Ice water thaw, 2016, 2016 Semen; the pH pH on tape 7.4, No motility Apr. 5, Plus 65 ul and and a Live 17/84 Dead 2016, of BTE − speed of stain. Percentage of sap Apr. 5, Fruc = ½% freezing needs to increase from 2016, Suc (pH need to 33%. Apr. 5, 7.4); Plus be Apr. 5, 2016 lost sample as 2016 65 ul BTE − changed. the caulk came out on Fruc + 1st Slow the thawing. Run Maple freeze Apr. 5, 2016 Ice water thaw Tree sap. down and and then palmed to warm 2015 (pH lower the because caulk came out. 7.23) pH to pH 7.5 on tape. No decrease motility. Live 8/92 Dead cell stain. metabolism. Apr. 5, 2016 Ice water thaw and then palmed to warm. pH 7.2. No motility. Live 6/94 Dead. 116 Apr. 4, Apr. 4, Odin None 40 ul of NO, NO, Apr. 4, 2016 Ice water thaw. 2016, 2016 Semen; NO, pH of 7. Live 0/100 Dead Apr. 4, Plus 40 ul Formula stain. No motility. 2016, of error Apr. 4, 2016 Ice water thaw. Apr. 4, Goshawk stopping pH of 7. Live 25/75 Dead 2016 Semen motility in stain. No motility. Extender = samples. Apr. 4, 2016 Ice water thaw. BTE − Fruc + The pH of 7. Live 9/91 Dead ½% addition stain. No motility. Suc w/pH of of 6.27.: glutathione Plus 80 ul and BTE − Fruc + NN- Amur Bis . . . sulfonic Maple + acid Glutathione + should NN- not have Bis . . . Sulfonic been Acid done. 117 Apr. 4, Apr. 4, Odin None 40 ul NO, NO, Apr. 4, 2016, Ice water thaw, 2016, 2016 Semen; NO, pH 7, No motility. Live Apr. 4, Plus 40 ul Formula 28/72 Dead. 2016, Goshawks error Apr. 4, 2016 Ice water thaw. Apr. 4, Extender stopping pH 7. No motility. Live 2016 (pH 6.27); motility in 0/100 Dead stain. Plus 80 ul samples. Apr. 4, 2016 Ice water thaw. of BTE − The pH 7, No motility. Sample Fruc + addition too small for a Live/Dead Amur of stain. 1 straw missing. Maple 1st glutathione run. + and Glutathione + NN- NN Bis . . . sulfonic Bis . . . sulfonic acid acid. should not have been done. 118 Apr. 5, Apr. 5, Odin None 33 ul Yes, Brix value Apr. 5, 2016 Ice water thaw, 2016, ( ) 2016 Semen; simple 2.5 pH on tape was 7, 15-20% Plus 66 ul addition moving initially. Live BTE − of sap 47/Dead 53. Longer Fructose + allowed acclimation may have Maple tree the cells increased survival. But sap tree #3 to longer acclimation 2015. Did survive. decreases survival in the not hen's seminal tubules acclimate when it does not have an with an extender added. extender. Only acclimated semen in its own tube and then added the sap. 119 Apr. 10, Apr. 5, Odin Back to the Yes, Yes Brix value Apr. 10, 2016, Ice water thaw, 2016, 2016 2015 2.5 pH 7 on test tape, About Apr. 11, Formulas. 5% vibrating in place, Live 2016, ( ). The 30/Dead 70. Would likely modification have done better if left that I above the vapors longer. made to Apr. 11, 2016 Ice water thaw, the pH was pH 7 on tape, Some stopping vibrating in place but not motility in moving forward, Live the cells so 31/Dead 69. It takes 90 I started seconds to process 1 back at sample into 4 tubes. baseline extenders. 45 ul Semen; Plus 45 ul of BTE − Fructose + ½% Sucrose (pH 7.51); Plus 90 ul of BTE − Fructose + Maple Tree #3 2015 (pH 6.48) 120 Apr. 10, Apr. 8, Odin Back to the Yes, the Apr. 10, 2016 Ice water thaw, 2016 2016 2015 original pH 7, 50% initially motile Formulas. 2015 sap to about 5% motile over The formulas about 10 minutes. Live modification support 62/Dead 38. It took 90 that I the cells seconds to package the 4 made to well. straws and this delay in the pH was getting it into the LN2 stopping likely allowed the osmotic motility in gradient across the cells to the cells so fade, allowing the cell to I started rehydrate prior to the back at freeze. This is conjecture, baseline but noted as a problem in extenders. references. Need a 45 ul processing time less than Semen, this. I stored a small then 45 ul sample of this tube in the of BTE − fridge from 7:30 AM to Fructose + 1:30 AM and more than ½% 75% were moving forward Sucrose and straight. (not frozen). (pH 7.51), then added 90 ul of BTE − Fructose + Maple Tree #3 2015 (p 121 Apr. 6, Odin Back to the Brix 2016 2015 value Formulas. 2.5 The modification that I made to the pH was stopping motility in the cells so I started back at baseline extenders. 55 ul Semen; Plus 55 ul of BTE − Fructose + ½% Sucrose (pH 7.51); Plus 100 ul of BTE − Fructose + Maple Tree #3 2015 (pH 6.48) 122 Apr. 7, Odin Back to the Brix 2016 2015 value Formulas. 2.5 The modification that I made to the pH was stopping motility in the cells so I started back at baseline extenders. 123 Apr. 8, Odin Back to the Brix 2016 2015 value Formulas. 2.5 The modification that I made to the pH was stopping motility in the cells so I started back at baseline extenders. 124 Apr. 7, Odin Back to the Brix 2015 2015 value Formulas. 2.5 The modification that I made to the pH was stopping motility in the cells so I started back at baseline extenders. 125 Apr. 8, Odin Back to the Brix 2016 2015 value Formulas. 2.5 The modification that I made to the pH was stopping motility in the cells so I started back at baseline extenders. 126 Apr. 10, Apr. 8, Odin Back to the Maybe, Brix Holder #4 trace sample 2016, 2016 2015 YES value because caulk came out of Apr. 11, Formulas. 2.5 capillary tube, Thawed in 2016 The Ice Water, No pH, No L/D modification stain. 10% moving forward that I with good motility.; made to Apr. 11, 2016 Ice water thaw, the pH was pH 7, 30% motile on visual stopping inspection, Live 50/Dead motility in 50. the cells so I started back at baseline extenders. 40 ul Semen; Plus 40 ul of BTE − Fructose + ½% Sucrose 2015 (pH 7.51); Plus 80 ul BTE − Fructose + Maple Tree sap #3 2015 (pH 6.48). 127 Apr. 10, Odin Back to the 2016 2015 Formulas. The modifications that I made to the pH was stopping motility in the cells so I started back at the baseline extenders. BIRCH sap STARTS HERE. 40 ul Semen, plus 40 ul of BTE − Fructose + 1st Run Alaska Birch (pH. 7.56), then added 40 ul BTE − Fructose + Maple Tree #3 2015 (pH. 6.48). 128 Apr. 10, Apr. 10, Odin 40 ul Yes Apr. 10, 2016 Ice water thaw, 2016 2016 Semen; pH 7 on tape, 25% moving Plus 40 ul straight forward, Live BTE − 57/43 Dead stain. Fructose + 1st run Alaska Birch (pH. 7.56); Plus 40 ul BTE − Fructose + Maple Tree sap tree #3 2015 (pH 6.48). How sample was Holder in tank it How storage Number frozen. pH of Diluent Diluent Type is located in Straw Type. straw performed. 4 Either flash frozen or 9.1 Diluent #1 Unknown. Unknown suspended above the liquid nitrogen for 30 seconds prior to immersion. 5 Flash frozen. 9.1 Diluent #1 2 Unknown 6 Either flash frozen or 9.1 Diluent #1 Unknown. Natellson This straw suspended above the Capillary for storage liquid nitrogen for 30 tube with 2 stored well seconds prior to caps. and did not immersion. explode. 7 Either flash frozen or 9.1 Diluent #1 Unknown. Unknown suspended above the liquid nitrogen for 30 seconds prior to immersion. 8 Flash frozen. 8.86 Diluent #2 2 Unknown 9 Either flash frozen or 8.86 Diluent #2 Unknown. Unknown suspended above the liquid nitrogen for 30 seconds prior to immersion. 10 Flash frozen. 8.86 Diluent #2 2 11 Either flash frozen or 8.86 Diluent #2 Unknown. Unknown suspended above the liquid nitrogen for 30 seconds prior to immersion. 12 Flash frozen. 8.86 Diluent #2 2 Unknown 13 Either flash frozen or 8.86 Diluent #2 Unknown. Unknown suspended above the liquid nitrogen for 30 seconds prior to immersion. 14 Either flash frozen or 9.08 Diluent #3 Unknown. Unknown suspended above the liquid nitrogen for 30 seconds prior to immersion. 15 Either flash frozen or Unknown None Unknown. Unknown suspended above the liquid nitrogen for 30 seconds prior to immersion. 16 Either flash frozen or 9.08 Diluent #3 Unknown. Unknown suspended above the liquid nitrogen for 30 seconds prior to immersion. 17 Flash frozen. 9.08 Diluent #3 3 Natellson This straw Capillary for storage tube with 2 stored well caps. and did not explode. 18 Flash frozen. 9.18 Diluent #4 2 Natellson Capillary tube with 2 caps. 19 Either flash frozen or 7.06 Trout Unknown. Natellson suspended above the Capillary liquid nitrogen for 30 tube with 2 seconds prior to caps. immersion. 20 Either flash frozen or 7.06 Trout Unknown. Natellson Exploded suspended above the Capillary and lost. liquid nitrogen for 30 tube with 2 seconds prior to caps. immersion. 21 Flash frozen. 7.06 Trout 2 Natellson Capillary tube with 2 caps. 22 Flash frozen. 7.06 Trout 5 Unknown 23 Suspended over liquid 7.16 Trout #2 Unknown. Natellson This straw nitrogen for 30 seconds Capillary for storage to exposed to liquid tube with 2 stored well nitrogen vapors and then caps. and did not flash frozen. explode. 24 Suspended over liquid 7.16 Trout #2 Floating Natellson Exploded nitrogen for 30 seconds on liquid Capillary on thaw to exposed to liquid nitrogen tube with 2 but did not nitrogen vapors and then and lost caps. lose the flash frozen. out of sample. holder. 25 Flash frozen. 7.16 Trout #2 5 Unknown 26 Flash frozen. 7.16 Trout #2 5 Unknown 27 Flash frozen. None 5 Unknown 28 Flash frozen. 7.16 Trout #2 5 Unknown 29 Suspended over liquid 7.14 Milk #1 Unknown. Natellson This straw nitrogen for 30 seconds Capillary for storage to exposed to liquid tube with 2 stored well nitrogen vapors and then caps. and did not flash frozen. explode. 30 Flash frozen. 7.42 Trout #2 5 Unknown plus Sorbitol and Arabogalactin. 31 10 mm of semen to 45 Turkey 6 Commercial Exploded mm of Turkey Extender, Extender semen and lost. Acclimated in the fridge plus DMA straw with for 30 minutes, Added 3 button ul (units) of DMA, Hung caps. in vapors for 10 minutes GREEN the placed in liquid ON TOP nitrogen. OF ALUMINUM HOLDER. 32 Semen acclimated in Turkey 6 Commercial FOUND fridge for 30 minutes, Extender semen FLOATING then DMA added, then plus (6%) straw with IN TANK hung over vapors for 10 DMA button MINUS minutes, then immersed caps. LABEL in liquid nitrogen. GREEN ON TOP OF ALUMINUM HOLDER. 33 Semen acclimated in Turkey 6 Commercial FOUND fridge for 30 minutes, Extender semen FLOATING then DMA added, then plus DMA straw with IN TANK hung over vapors for 10 button MINUS minutes, then immersed caps. LABEL in liquid nitrogen. GREEN ON TOP OF ALUMINUM HOLDER. 34 Semen acclimated in Turkey 6 Commercial FOUND fridge for 30 minutes, Extender semen FLOATING then DMA added, then plus DMA straw with IN TANK hung over vapors for 10 button MINUS minutes, then immersed caps. LABEL in liquid nitrogen. GREEN ON TOP OF ALUMINUM HOLDER. 35 Semen acclimated in Turkey 6 Commercial Exploded fridge for 30 minutes, Extender semen and not then DMA added, then plus DMA straw with lost. But hung over vapors for 10 button explosion minutes, then immersed caps. was loud in liquid nitrogen. GREEN and semen ON TOP cells likely OF damaged ALUMINUM due to HOLDER. trauma. I am guessing that this sample went to Juniper because 4 were found floating free on top of the liquid nitrogen and not labeled. 36 See prior note Turkey 6 Unknown Extender plus DMA 37 Flash frozen. Turkey 5 Commercial Exploded Extender semen and lost. plus DMA straw with button caps. RED ON TOP OF ALUMINUM HOLDER 38 Flash frozen. Turkey 5 Commercial Exploded Extender semen and lost. plus straw with (10%) button DMA caps. RED ON TOP OF ALUMINUM HOLDER 39 Flash frozen. Turkey 5 Commercial Exploded Extender semen and lost. plus straw with (10%) button DMA caps. RED ON TOP OF ALUMINUM HOLDER 40 Flash frozen. Turkey 5 Commercial Exploded Extender semen and lost. plus straw with The (18%) button reinforced DMA caps. RED plastic ON TOP coated OF glass ALUMINUM capillary HOLDER tubes withstand explosions. 41 SEE NOTES ON 2013 Turkey SAMPLES. Extender plus DMA 42 In fridge 10 minutes to 6.74 Turkey #2 #42, white acclimate, plus 1.5 units Extender straw, DMA, above vapors for plus DMA, Natellson 10 minutes, immersed 5% DMA tube, suddenly into LN2 capped on 1 end firmly and outside end not capped. 43 Acclimated in fridge 10 6.74 Turkey #2 #43 White minutes, plus 1.5 units Extender straw, DMA (5%), hung over plus DMA, standard vapors for 10 minutes 5% DMA capillary within 1 minute of adding tube, plus DMA, dunked into LN2. two crito caps and 1 Natellson cap. 44 Acclimated in fridge 10 6.74 Turkey #2 #44 White Straw minutes, plus 3 ul of Extender straw, exploded DMA (6%), Acclimated in plus 6% standard on thaw, fridge for 10 minutes, DMA capillary but one suspended above the tube plus 2 end stayed vapors 10 minutes, then critocaps closed. dunked in LN2. and 1 Natellson cap. 45 Acclimated in fridge 10 6.74 Turkey #2 #45 White minutes, plus 2 ul of extender straw, DMA (10%), hung over plus 10% Standard vapors for 10 minutes, DMA capillary plunged into LN2 tube, plus 2 caps on one end and clay on the far end. 46 Acclimated in fridge (35 6.74 Turkey #2 #46 White F.), Plus 2 ul of DMA, extender straw, tube hung over vapors for 10 plus 10% type not minutes, plunged into DMA recorded. LN2. 47 Acclimated in fridge at 35 6.74 Turkey #2 #47, two F. for 10 minutes, one Extender tubes, sample hung over vapors plus 6% White for 7 minutes and the DMA straw, other 10 minutes, and Natellson then dunked into LN2. tube, plus clay and Natellson cap on pointed end, and Natellson cap on top end. 48 Acclimated in the fridge 6.74 Turkey #2 White at 35 F., 1.5 ul of DMA extender straw, added, over vapors 10 plus 7% Natellson minutes, dunked into DMA. capillary LN2. tube plus clay in both ends and Natellson cap on pointed end. 49 Acclimated in fridge at 6.74 Turkey #2 White 35 F. for 10 minutes, plus Extender straw, 3 ul of DMA, hung over plus 6% Natellson vapors 10 minutes, DMA tube plus plunged into LN2. clay on both ends and rubber cap on pointed end. 50 Acclimated in fridge at 6.74 Turkey #2 White 35 F. for 10 minutes, plus extender straw, 75 ul 2 ul of DMA (7%), over plus 7% standard vapors for 10 minutes, DMA. capillary then plunges into LN2. tube plus 1 critocap and Natellson Cap on one end and Clay on the other end. 51 Acclimated in the fridge 6.74 Turkey #2 White at 35 F., 1.25 (5%) DMA extender straw, 75 ul added, hung over vapors with 5% standard for 10 minutes, plunged DMA. capillary into LN2. tube plus one end a critocap and one end a Natellson cap. 52 Acclimated in the fridge 6.74 Turkey #2 White at 35 F., 2.7 ul of DMA extender straw, added, Hung over vapors with 6% Natellson 10 minutes, plunged into DMA. Capillary LN2. tube, plus clay on both ends, and Natellson cap on pointed end. 53 Acclimated in the fridge 6.74 Turkey #2 White at 35 F. for 15 minutes, extender straw, 2.23 ul of DMA added, with 6% Natellson hung over vapors 10 DMA. Capillary minutes, plunged into tube, plus LN2. clay on both ends, and Natellson cap on pointed end. 54 Acclimated in the fridge 6.74 Turkey #2 White at 35 F. for 15 minutes, extender straw, plus 1 ul of DMA (5%), with 6% Standard hung over vapors 10 DMA. 75 ul minutes, plunged into capillary LN2. tube, clay on one end and Natellson cap and critocap on other end. 55 Acclimated in the fridge 6.74 Turkey #2 White at 35 F. for 15 minutes, extender straw, added 2.5 ul DMA, hung with 5% Natellson over vapors 10 minutes, DMA. tube plus plunged into LN2. clay on pointed end and Natellson caps on both ends. 56 Acclimated in the fridge 6.74 Turkey #2 Red Straw, for 15 minutes, plus 1 ul extender, Natellson of DMA (5%), hung over with tube, with vapors for 10 minutes, Maple clay and plunged into LN2. Syrup, Natellson with 5% cap on DMA. pointed end and just clay on large end. 57 Acclimated in the fridge Turkey #4 holder Red straw. The caps for 10 minutes, plus 1 ul Extender in Tank 2 This was a exploded of DMA (5%), dropped by itself 75 ul mylar off of the into the LN2. coated ends and capillary most of the tube, sample caulked on was lost. both ends, plus 2 critocaps, plus a teal cap on the semen end. 58 Acclimated in the fridge Beltsville #4 Holder Red soda Sample for 15 minutes, plus 3 ul Turkey in Second straw, with explodes of DMA Extender tank. a 75 ul across the plus .2 mg Mylar garage. Inositol to coated 10 ml of capillary Extender. tube, caulked on both ends, with two critocaps, and one teal cap. Plus aluminum holder. 59 Acclimated in the fridge Beltsville #4 Holder Red soda Tube for 15 minutes, Plus 2.6 ul Turkey in the straw, with performed of DMA and then flash Extender Second a 75 ul well. frozen. plus .2 mg tank Mylar Inositol to coated 10 ml of capillary Extender. tube, caulked on both ends, with two critocaps, and one teal cap. With Aluminum holder. 60 Acclimated in the fridge Beltsville #4 Holder Red soda Exploded for 15 minutes and then Turkey in the straw, with but kept flash frozen Extender Second a 75 ul sample. plus .2 mg tank Mylar Inositol to coated 10 ml of capillary Extender. tube, caulked on both ends, with two critocaps, and one teal cap, With Aluminum holder. 61 Acclimated in the fridge Beltsville #3 Holder, Red soda Straw for 15 minutes and then Turkey Tank 2 straw, with performed flash frozen Extender a 75 ul well. plus .2 mg Mylar Inositol to coated 10 ml of capillary Extender. tube, caulked on both ends, with two critocaps, and one teal cap, 62 Acclimated in the fridge Beltsville #3 Holder, Red soda Straw for 15 minutes and then Turkey Tank 2 straw, with exploded flash frozen Extender a 75 ul on thaw, plus .2 mg Mylar but sample Inositol to coated was 10 ml of capillary preserved. Extender. tube, caulked on both ends, with two critocaps, and one teal cap. 63 Acclimated in the fridge Beltsville #3 Holder, White soda Tube for 15 minutes and then Turkey Tank 2 straw, with performed flash frozen Extender a Mylar well. plus .2 mg coated Inositol to capillary 10 ml of tube, Extender. caulked on both ends, with two critocaps, and one teal cap. 64 Acclimated in the fridge Beltsville #3 Holder, Red soda Tube for 15 minutes and then Turkey Tank 2 straw, with exploded, flash frozen Extender a 75 ul Lost most plus .2 mg Mylar of the Inositol to coated sample. 10 ml of capillary Extender. tube, caulked on both ends, with two critocaps, and one teal cap. 65 Acclimated in the fridge Beltsville #3 Holder, Red soda Tube for 15 minutes and then Turkey Tank 2 straw, with performed flash frozen Extender a 75 ul well. plus .2 mg Mylar Inositol to coated 10 ml of capillary Extender. tube, caulked on both ends, with two critocaps, and one teal cap. 66 Acclimated in the fridge Beltsville #3 Holder, White soda Straw for 15 minutes and then Turkey Tank 2 straw, with empty on flash frozen Extender a Mylar thaw. plus .2 mg coated Inositol to capillary 10 ml of tube, Extender. caulked on both ends, with two critocaps, and one teal cap, No sample in tube on thaw. 67 No acclimation. Flash SAP #2 Holder Orange Trace Frozen. Beltsville Tank 2 soda straw, sample left Turkey with 75 ul on thaw Extender capillary due to minus the tube, plus exploding. fructose, clay both plus ends, plus Maple critocaps. Tree sap, first run, Tree number 3. 68 No acclimation, flash Likely #3 Holder, Red soda Straw frozen. same as Tank 2 straw, in performed samples Aluminum well. Did above sleeve, with not and below 75 ul mylar explode. this line. capillary tube, w/ clay and critocap with teal cap 1 end, and clay and crito cap other end. 69 No acclimation was done SAP, #2 holder, Aluminum This straw and it was flash frozen. It BTE, no Tank 2. sleeve with did well was carried to the garage fructose, a White and did not on cold gel packs. plus soda straw, explode, Maple with 75 ul but the Tree sap, capillary large teal first run, tube, 1 end caps on Tree left open, the end is number 3. other end slow to capped thaw. The with clay, 1 open end critocap, 1 is key to teal cap. this success. 70 No acclimation was done SAP, #2 holder, Red soda Straw and it was flash frozen. It BTE, no Tank 2. straw with performed was carried to the garage fructose, holes, plus well, did on cold gel packs. plus plastic not Maple poultry explode. Tree sap, straw, with first run, a 75 ul Tree capillary number 3. tube, 1 end left open, the other end is clayed, slid into poultry straw with cotton facing down. Straw is ventilated. 71 No acclimation was done SAP, #2 holder, Red soda and it was flash frozen. It BTE, no Tank 2. straw with was carried to the garage fructose, holes, plus on cold gel packs. plus plastic Maple poultry Tree sap, straw, with first run, a 75 ul Tree capillary number 3. tube, 1 end left open, the other end is clayed, slid into poultry straw with cotton facing down. 72 No acclimation was done SAP, #2 holder, Red soda Straw and it was flash frozen. It BTE, no Tank 2. straw with performed was carried to the garage fructose, holes, plus well. on cold gel packs. plus plastic Maple poultry Tree sap, straw, with first run, a 75 ul Tree capillary number 3. tube, 1 end left open, the other end is clayed, slid into poultry straw with cotton facing down. 73 No acclimation was done SAP, #1 holder, Pink soda Straw and it was flash frozen. BTE, no Tank #2 straw, plus performed fructose, a plastic well. It did plus poultry not BIRCH straw, plus explode. tree sap. a 75 ul Sucrose capillary is lower tube with 1 than end sealed Maple with clay, tree sap. with cotton on poultry straw facing down. Number on straw reads 67. 74 2 samples were 16% MA Holder #3 Orange produced due to the used as in Can #1. (#68 on volume of the sample. half of the straw) and Both were flash frozen. sample so Pink soda No gel packs were used. the final straws (# concentration 68 on of the straw), with MA was a plastic 8%. poultry straw inside, with a 75 ul mylar capillary tube clayed on one end, with cotton on poultry straw facing down. 75 Separated into 3 straws 16% MA #3 Holder 3 green as it was so large. On gel used as Tank #1. soda packs less than 2 half of the straws, no minutes and then flash sample so holes cut in frozen. the final them. concentration Plastic of the poultry MA was straws with 8%. 75 ul mylar capillary tubes, 1 end has clay, the cotton on the poultry straw faces down. 76 Acclimated in the fridge 7.5 on pH Beltsville #3 Holder Soda for 10 minutes, DMA tape after Turkey Tank #1. straw plus added, used bottom of thaw. Extender, plastic fridge at 40 F. AND no poultry TURNED DOWN THE fructose, straw, plus FRIDGE FOR THE plus ½% a 75 ul FIRST TIME so it went sucrose. mylar up in temperature. THE capillary TOP OF THE FRIDGE tube with WAS AT 35 F. AND THE clay on 1 BOTTOM OF THE end. With FRIDGE WAS AT 40 F. cotton on BEFORE I TURNED IT poultry DOWN. It now sits at 42 straw at the bottom where I facing am now holding the down. Not samples so it is 9 ventilated. degrees warmed for Blue in processing. The color. samples done from here out are done at a warmer temperature. 77 Flash frozen after 10 7.5 pH on BTE, no Holder #3 3 soda minutes of acclimation at pH tape fructose, in Can #1. straws, 41 F. after plus ½% light green, thaw. sucrose, dark green, and DMA orange, with a poultry straw inside that and a 75 ul mylar tube inside of that. Green straw was not ventilated and the Orange straw was not ventilated. 78 Flash frozen after 10 BTE, no Holder #3 2 soda Storage minutes of acclimation at fructose, in Can #2. straws, with a 41 F. plus Birch both pink, Ventilated Tree sap. both soda straw, ventilated, poultry with poultry straw straw and a inside that 75 ul mylar [Poultry capillary straw tube inside crimped on that. Clay top end on one end and cotton leaving it on the open on bottom top. end], with a 75 ul mylar coated capillary tube, clay on one end only with the other end left open to LN2; Performed the BEST Keep the material open so they do not explode. All samples stored this way from this point on. 79 Sample was not Birch tree Holder #3, 2 Soda Well, no acclimated in the fridge. sap in Tank #2. straws, one problems. It was mixed, placed in BTE, No yellow and tubes, and then put fructose one green, between gel packs from both the fridge, carried out to ventilated the LN2 can, and flash with holes; frozen. with a poultry straw, cotton facing down, crimped on top end; with 75 ul capillary tubes, clayed on one end. 80 Acclimated in the fridge Maple Holder #3, 2 soda Well, no for 10 minutes in tree sap Tank #2. straws, one problems. separate tubes, then in BTE, orange and combined and packaged, no one pink, then flash frozen fructose. both ventilated, with poultry straw inside, with 75 ul mylar coated capillary tube inside. 81 Acclimated for 10 Maple Holder #3, 2 Soda Well, no minutes in the fridge at tree sap Tank #2. straws that problems. 41 F., and then flash in BTE, were frozen no ventilated, fructose. with poultry straw inside, cotton end down; 75 ul mylar capillary tube, clay on one end 82 Semen and BTE − Fruc, + pH on pH Birch tree 2 straws 4 green Does not ½% sucrose tape after sap in in Holder soda explode acclimated in separate thaw was BTE, No #6 tank 1, straws that but thaws tube from Birch sap BTE, 7.0, 7.2, fructose and 2 were too slowly. at 42 F. in fridge for 10 7.0, 7.5. straws in ventilated, minutes, combined, then Holder #5 75 ul mylar packaged in 4 tubes, 75 tank 1. capillary ul capillary tube, caulked tube, inside one end; placed in poultry poultry straw, cotton straw, down; inside ventilated inside soda straw. 4 straws ventilated made. All green soda soda straw. straws. 83 Semen and BTE − Fruc, + pH on pH Maple 2 straws 3 yellow Good ½% sucrose tape after tree sap in Holder soda acclimated in separate thaw was in BTE, #6 tank 1, straws that tube from Maple tree sap 7.5-8, 7.0, 7.0 no and 1 were BTE, at 42 F. in fridge for fructose. straws in ventilated, 10 minutes, combined, Holder #5 75 ul mylar then packaged in 4 tank 1. capillary tubes, 75 ul capillary tube, inside tube, caulked one end; poultry placed in poultry straw, straw, cotton down; inside inside ventilated soda straw. 3 ventilated straws made. 3 Yellow soda straw. straws. 84 Semen and BTE − Fruc, + Mar. 1, 2016 Maple 2 straws 3 pink soda Tubes ½% sucrose pH on tree sap in Holder straws that store well acclimated in separate tape of 7. in BTE, #6 tank 1, were but it is tube from Maple tree sap Mar. 6, 2016 no and 1 ventilated, best to use BTE, at 44 F. in fridge for pH on fructose. straws in 75 ul mylar the soda 10 minutes, combined, tape of 7.5. Holder #5 capillary straw and then packaged in 3 tank 1. tube, inside the mylar tubes, 75 ul capillary poultry capillary tube, caulked one end; straw, tube only. placed in poultry straw, inside cotton down; inside ventilated ventilated soda straw. 3 soda straw. straws made. 3 Pink One straws. sample was a capillary tube and soda straw only. 85 Semen and BTE − Fruc, + pH on pH Maple 2 straws 3 Yellow ½% sucrose tape was tree sap in Holder soda acclimated in separate 7.0, pH on in BTE, #6 tank 1, straws that tube from Maple tree sap pH tape no and 1 were BTE, at 44 F. in fridge for was 7.0 fructose/ straws in ventilated, 10 minutes, combined, with 10% Holder #5 75 ul mylar then packaged in 3 Yolk. tank 1. capillary tubes, 75 ul capillary tube, inside tube, caulked one end; poultry placed in poultry straw, straw, cotton down; inside inside ventilated soda straw. 3 ventilated straws made. 3 Yellow soda straw. straws. 10% Yolk Added to both mixes. 86 All volumes added Maple Holder #5, 1 pink soda together, No acclimation. tree sap Tank 1. straw that Flash frozen. 10% Yolk in BTE, was Added. no ventilated, fructose/ 75 ul mylar with 10% capillary Yolk. tube, inside a poultry straw, inside the soda straw. 87 Acclimated for 10 pH of 7 on Maple Holder #6, Unknown Stores minutes in the fridge at pH tape tree sap in can #1. color of well, but 43 F., and then flash after in BTE no soda straw. thaws too frozen. 10% Yolk thaw. fructose/ Was a Pink slow. added. with yolk. ventilated soda straw. 88 Acclimated for 10 2016 pH Maple Holder #6, Two pink Straws minutes in the fridge at on pH tree sap in can #1 soda work well 43 F., and then flash tape 7, pH in BTE no has 2 pink straws, but insulate frozen. 10% Yolk on pH fructose/ straws, ventilated, too well on added. tape of 7 with yolk. and with a 75 ul thawing. Holder #5, mylar in can #1 capillary has 1 pink tube inside straw. a poultry straw. 89 Acclimated for 10 2016 pH Birch tree Holder #6 Two Does not minutes in the fridge at on tape sap in has 1 orange explode 43 F., and then flash of 7.0 BTE, No orange soda but thaws frozen. 10% Yolk fructose, soda straws, too slowly. added. Plus straw, and ventilated, YOLK Holder #5 with 75 ul has 1 mylar orange capillary soda tubes straw. inside poultry straws. 90 Acclimated for 10 pH on Birch tree Holder #6 Three Does not minutes in the fridge at tape of sap in has 2 green soda explode 43 F., and then flash 7.0, 7.0, BTE, no green straws, but thaws frozen. 10% Yolk 7.0. fructose soda ventilated, too slowly. added. Plus Yolk. straws with 75 ul and mylar Holder #5 capillary has 1 tubes green inside soda poultry straw. straws. 91 Temperature dropped pH on MA only Holder #4, 2 soda only by using gel packs. tape after Tank 1. 2 straws, one thaw was straws, green and 7.5, 7.5 one is one deep green and blue. the other Ventilated deep with 75 ul purple. mylar capillary tube inside a poultry straw. 92 Temperature dropped MA only Holder 3, 1 green Stores only by using gel packs. Tank 1, 1 soda straw, well, but green ventilated. thaws too soda With 75 ul slow. straw. mylar capillary tube inside a poultry straw. 93 Chilled at 45 F. for 10 pH on MA only Holder 4, 3 green minutes and then flash tape after Tank 1, 3 soda frozen. thaw was green straws, 7.5, 7.5, 7.5. soda ventilated, straws all with 75 ul here. mylar capillary tube inside a poultry straw. 94 Chilled at 45 F. for 10 DMA Holder #5, 1 orange minutes and then flash Tank 1. soda straw frozen. with 2 poultry straws inside of it because I ran out of staples and had 1 soda straw left with a staple in it, marked with # 89 on it. 95 It was mixed at room 2016 pH 7 Purdy Holder 5 2 orange Ventilated temperature with no 10% and 6, soda soda straw acclimation and then Maple Tank 1. straws with with poultry flash frozen. Lack of Tree Sap, 75 ul mylar straw and acclimation reduces cell 326 capillary mylar survival. mOsm. tubes capillary inside tube inside poultry that thaws straws. too slowly. 96 It was mixed at room Purdy Holder # 1 light temperature with no 10% 5, Tank 1, yellow soda acclimation and then Maple Light straw with flash frozen. Tree Sap, yellow 75 ul mylar 326 soda capillary mOsm. straw. tube inside a poultry straw. 97 It was mixed at room pH of 7 on Purdy Holder # 1 pink soda Stores temperature, sandwiched pH tape 10% 6, Tank 1. straw that well, but between gel packs at 43 after Maple Pink soda was thaws too F., and then flash frozen. thaw. Tree Sap, straw. ventilated, slow. 326 75 ul mylar mOsm. capillary tube, inside a poultry straw, inside the soda straw. 98 Purdy formulas begin with # 95. 99 It was mixed at room Feb. 24, 2016 Purdy 20% Holder #4 3 pink soda temperature, sandwiched Pink Maple has 2 pink straws that between gel packs at 43 straw, no Tree Sap, soda are F., and then flash frozen. pH done, 308 straws ventilated, Second mOsm. and 75 ul mylar straw pH Holder #5 capillary of 7.5, has 1 pink tube, inside Last straw soda a poultry no pH straws. straw, done. inside the soda straw. 100 It was mixed at room Mar. 6, 2016 Purdy 20% Three 4 greens Good, temperature, sandwiched pH 7 on Maple green soda stores well between gel packs at 43 tape after Tree Sap, soda straws that but thawed F., and then flash frozen. thaw. 308 straws in are too slow. Mar. 6, 2016 mOsm. Holder #6, ventilated, pH of 7 on 1 straw in 75 ul mylar tape after holder #5. capillary thaw. tube, inside a poultry straw, inside the soda straw. 101 It was mixed at room Mar. 2, 2016 Purdy Two 2 Yellow Soda straw temperature, sandwiched pH of 7 on 10% yellow soda did not between gel packs at 43 pH tape Maple straws, straws that have F. for 2 minutes, and then after tree sap one in are capillary flash frozen. thaw. holder #6, ventilated, tube in it, and one 75 ul mylar Lost in the in holder capillary tank. #5. tube, inside a poultry straw, inside a soda straw. 102 It was mixed at room Mar. 2, 2016 Purdy 5% 3 orange 3 orange Good, temperature, sandwiched pH of 7; Maple soda soda stores well between gel packs at 43 Mar. 6, 2016 tree sap. straws, 2 straws that but thawed F. for 2 minutes, and then pH of 7. in holder are too slow. flash frozen. No #6, and 1 ventilated, acclimation. in holder 75 ul #5 capillary tube inside a poultry straw. 103 It was mixed at room 7.5 and 7, Purdy 4 green 4 green Good temperature, with no 7, 7. 10% soda soda acclimation, and then Maple straws, 2 straws that flash frozen. tree sap, straws in were plus 5% holder #5 ventilated, (13 ul) of and 2 75 ul mylar DMA. straws in capillary holder #6. tube, inside poultry straw, inside ventilated soda straw. 104 It was mixed at room 7 Purdy Yellow Yellow Good temperature, with no 10% straw in soda straw acclimation, and then Maple holder #5 that was flash frozen. tree sap ventilated, plus 75 ul mylar Arabogalactin capillary and tube, inside then 12% poultry MA. straw, inside ventilated soda straw. 105 Chilled 15 minutes and 7.51 and 7.23. 2015 2 straws Mylar Stores well then second diluent (adjusted extenders in Holder capillary and thaws added. Packaged quickly wrong that have #6, 1 tube, well. and then flash frozen. and bad sap in straw in caulked on for them. Holder #5 one end, motility). Second inside a diluent small had pH ventilated adjusted soda straw. up with bicarb. 106 Acclimated in the fridge 7.51 and 7.23. 2015 2 straws Mylar Stores well at 42 F. for 15 minutes (adjusted extenders in Holder capillary and thaws and then flash frozen. wrong that have #6 and 1 tube, well. and bad sap in straw in caulked on for them. Holder #5. one end, motility). Second inside a diluent small had pH ventilated adjusted soda straw. up with bicarb. 107 Acclimated 16 minutes at pH 7.51 2015 2 straws Mylar Stores well 42 F. and then packages then extenders in Holder capillary and thaws into 3 straws and flash pH 7.23 that have #6 and 1 tube, well. frozen. (adjusted sap in straw in caulked on wrong them. Holder #5. one end, and bad Second inside a for diluent small motility). had pH ventilated adjusted soda straw. up with bicarb. 108 Acclimated 16 minutes at pH 7.51 2015 2 straws Mylar Stores well 42 F. and then packages then extenders in Holder capillary and thaws into 3 straws and flash pH 7.23 that have #6 and 1 tube well. frozen. (adjusted sap in straw in caulked wrong them. Holder #5. one end and bad Second with for diluent ventilated motility). had pH soda straw. adjusted up with bicarb. 109 Acclimated 16 minutes at pH of 7.51 2015 2 straws Mylar Stores well 42 F. and then packages then pH extenders in Holder capillary and thaws into 4 straws and flash of 7.23 that have #6 and 2 tube well. frozen. (adjusted sap in straws in caulked wrong them. Holder #5. one end and bad Second with for diluent ventilated motility). had pH soda straw. adjusted up with bicarb. 110 Acclimated 16 minutes in pH of 7.51 2015 2 straws Mylar Stores well the fridge at 42 F. and then pH extenders in Holder capillary and thaws then flash frozen. of 7.23 that have #6 and 1 tube well. (adjusted sap in straw in caulked wrong them. Holder #5. one end and bad Second with for diluent ventilated motility). had pH soda straw. adjusted up with bicarb. 111 Acclimated for 18 pH of 7.51 2015 2 straws Mylar Stores well minutes at 42 F. in the then pH extenders are in capillary and thaws fridge and then flash of 7.23 that have Holder #6 tube well. frozen. (adjusted sap in and 1 caulked wrong them. straw is in one end and bad Second Holder #5. with for diluent ventilated motility). had pH soda straw. adjusted up with bicarb. 112 Acclimated on gel packs 7.4 then BTE 1 straw in Mylar Caulk at 42 F. for 16 minutes. 7.23 both minus holder #5 capillary tends to Then flash frozen. One 2015 diluents fructose, and 2 tube come out sample got caught on the with sap plus ½% straws in caulked on thaw holder and stayed above that both sucrose holder #4. one end due to LN2 the LN2 and was not had pH with pH with pressure flash frozen. The other adjustments adjusted ventilated inside the samples in the group that with soda straw. capillary were flash frozen and went bad. bicarb tube. died. from 7.51 to 7.4.; Then added BTE − Fructose + Maple Tree Sap tree #3 with pH adjusted with bicarb to 7.23. 113 Acclimated for 16 7.4 then BTE 1 straw in Mylar Stores well minutes in the fridge at 7.23 both minus holder #5 capillary and thaws 42 F. and then flash 2015 diluents fructose, and 2 tube well. frozen. with sap plus ½% straws in caulked that both holder #4. one end had pH sucrose with adjustments with pH ventilated that adjusted soda straw. went bad. with bicarb from 7.51 to 7.4.; Then added BTE − Fructose + Maple Tree Sap tree #3 with pH adjusted with bicarb to 7.23. 114 Acclimated for 16 7.4 then BTE 1 tube in Mylar minutes in the fridge at 7.23 both minus Holder #5 capillary 42 F. and then flash 2015 diluents fructose, and 1 tube frozen. with sap plus ½% tube in caulked that both sucrose Holder #4. one end had pH with pH with adjustments adjusted ventilated that with soda straw. went bad. bicarb from 7.51 to 7.4.; Then added BTE − Fructose + Maple Tree Sap tree #3 with pH adjusted with bicarb to 7.23. 115 Acclimated 20 minutes and then flash frozen. 116 Acclimated 15 minutes pH 6.27 Amur 3 straws Mylar Stores well and then flash frozen. then Maple + in Holder capillary and thaws pH 6.74. BTE − #3. tube well. Fruc with caulked adjusted one end pH of 6.74 with with ventilated Glutathione + soda straw. NN- Bis . . . Sulfonic Acid. 117 Acclimated 15 minutes pH 6.27 Amur 4 straws Mylar Stores well and then flash frozen. then Maple + in Holder capillary and thaws pH 6.74. BTE − #3. tube well. Fruc with caulked adjusted one end pH of 6.74 with with ventilated Glutathione + soda straw. NN- Bis . . . Sulfonic Acid. 118 Acclimated semen in its pH 6.48 BTE − 2 straws Mylar own tube for 5 minutes Fructose + in holder capillary and then added in the Maple #3. tube + Maple tree sap tree #3 Tree #3 Critocap + (2015). Then slowly sap, Blue cap + lowered into LN2. 2015. Ventilated (original soda straw. 6.48 pH from 2015) 119 Put in holder #5 above 7.51. then 6.48 BTE − #5 Mylar Caulk liquid nitrogen vapors for Fructose + capillary tends to 10 seconds and then ½% tube, come out flash froze with slow Sucrose caulked on on thaw immersion. (pH 7.51), one end, due to LN2 then BTE − inside a pressure Fructose + small inside the Maple ventilated capillary Tree #3 soda straw. tube. sap, 2015. (pH 6.48). 120 Acclimated 15 minutes at 7.51 then 6.48 BTE − 4 straws Mylar Caulk 42 F. then hung over the Fructose + in Holder capillary tends to vapors 15 seconds and ½% #3. tube, come out then slowly lowered into Sucrose caulked on on thaw LN2. (pH 7.51); one end, due to LN2 then BTE − inside a pressure Fructose + small inside the Maple ventilated capillary Tree #3 soda straw. tube. sap, 2015. (pH 6.48). 121 Acclimated 15 minutes at 7.51 then 6.48 BTE − 2 straw in Natellson 42 F. then hung over the Fructose + Holder #3. Capillary vapors 15 seconds and ½% tube with 1 then slowly lowered into Sucrose cap in a LN2. (pH 7.51); large then BTE − ventilated Fructose soda straw. plus (Orange). Maple Tree sap #3 2015 (pH. 6.48) 122 Acclimated 15 minutes at pH 7.51 BTE − 1 straw in Natellson 42 F. then hung over the then Fruc + Holder #3. capillary vapors 15 seconds and pH 6.48. ½% Suc tube + cap + then slowly lowered into (pH 7.51); Large LN2. then BTE − ventilated Fruc + soda straw 1st Run (Green). Maple tree sap. 2015 123 124 125 126 Acclimated for 15 pH 7.51 BTE − Three Mylar Caulk minutes at 42 F. in the then Fructose + straws in capillary tends to fridge, and then flash pH 6.48. ½% Holder #5, tube come out frozen in LN2. Sucrose 1 Straw in caulked on thaw 2015 Holder #4. one end due to LN2 (pH 7.51); with pressure then ventilated inside the added soda straw. capillary BTE − tube. Fructose + Maple Tree Sap tree #3 2015 (pH 6.48) 127 pH 7.56 #4 Mylar then capillary pH 6.48 tube caulked one end with ventilated soda straw. 128 Acclimated for 15 pH 7.56 BTE − 3 straws Mylar Caulk minutes then suspended then Fructose in Holder capillary tends to over LN2 vapors for 15 pH 6.48 plus 1st #4. tube come out seconds and then slowly run caulked on thaw lowered into LN2. Alaska one end due to LN2 Birch with pressure (pH 7.56) ventilated inside the and then soda straw. capillary BTE − tube. Fructose + Maple Tree #3 2015 (pH 6.48) 

I claim:
 1. A method of cryogenically preserving sperm comprising: a. combining sperm to be cryogenically preserved and a composition that comprises (1) a cryoprotectant, comprising one or more tree saps; and (2) an extender medium to produce a sperm/medium combination; and b. subjecting the combination to conditions that result in cryopreservation of sperm, thereby producing a cryopreserved combination that comprises cryopreserved sperm, wherein the one or more tree saps is derived from cold-hardy trees.
 2. The method of claim 1 wherein the cryopreserved sperm of step (b) demonstrates survival greater than 50% after thawing.
 3. The method of claim 1 wherein the cryopreserved sperm of step (b) demonstrates motility greater than 30% after thawing.
 4. The method of claim 1 wherein the one of more tree saps is the only cryoprotectant.
 5. The method of claim 1 wherein an additional cryoprotectant is added.
 6. The method of claim 1 wherein the sperm is avian sperm.
 7. The method of claim 6 wherein the sperm is derived from the Northern goshawk (Accipiter gentilis).
 8. The method of claim 1 wherein the sperm is derived from a non-human mammal.
 9. The method of claim 8 wherein the sperm is derived from an animal type selected from the group consisting of canine, avian, cattle, porcine and equine.
 10. The method of claim 1 wherein the one or more tree saps is a first run sap.
 11. The method of claim 1 wherein the extender medium does not contain fructose.
 12. The method of claim 1 wherein the method comprises the additional step of subjecting the combination to a temperature between −80° C. and −198° C. for a period of at least one day.
 13. The cryopreserved combination resulting from the method of claim
 1. 14. The cryopreserved combination resulting from the method of claim
 2. 15. The cryopreserved combination resulting from the method of claim
 3. 16. A method of fertilizing an egg cell comprising the step of thawing a cryopreserved combination produced by the method of claim 1, and introducing the combination to an unfertilized egg cell, wherein the egg cell becomes fertilized.
 17. The method of claim 16, wherein the egg cell is an avian egg.
 18. The method of claim 16, wherein the egg cell is a mammalian egg.
 19. A composition comprising tree sap, semen, and extender medium, wherein the tree sap is a cold-hardy tree sap.
 20. The composition of claim 19, wherein the sap is at least 50% by volume of the composition. 